Grapevine leafroll virus (type 2) proteins and their uses

ABSTRACT

The present invention relates to isolated proteins or polypeptides of grapevine leafroll virus (type 2). The encoding DNA molecules either alone in isolated form or in an expression system, a host cell, or a transgenic grape plant are also disclosed. Other aspects of the present invention relates to a method of imparting grapevine leafroll resistance to grape and tobacco plants by transforming them with the DNA molecules of the present invention, a method of imparting beet yellows virus resistance to a beet plant, a method of imparting tristeza virus resistance to a citrus plant, and a method of detecting the presence of a grapevine leafroll virus, such as GRLaV-2, in a sample.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/047,194, filed May 20, 1997.

This work was supported by the U.S. Department of AgricultureCooperative Grant No. 58-2349-9-01. The U.S. Government may have certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to grapevine leafroll virus (type 2)proteins, DNA molecules encoding these proteins, and their uses.

BACKGROUND OF THE INVENTION

The world's most widely grown fruit crop, the grape (Vitis sp.), iscultivated on all continents except Antarctica. However, major grapeproduction centers are in European countries (including Italy, Spain,and France), which constitute about 70% of the world grape production(Mullins et al., Biology of the Grapevine, Cambridge, U.K.:UniversityPress (1992)). The United States, with 300,000 hectares of grapevines,is the eighth largest grape grower in the world. Although grapes havemany uses, a major portion of grape production (˜80%) is used for wineproduction. Unlike cereal crops, most of the world's vineyards areplanted with traditional grapevine cultivars, which have beenperpetuated for centuries by vegetative propagation. Several importantgrapevine virus and virus-like diseases, such as grapevine leafroll,corky bark, and Rupestris stem pitting, are transmitted and spreadthrough the use of infected vegetatively propagated materials. Thus,propagation of certified, virus-free materials is one of the mostimportant disease control measures. Traditional breeding for diseaseresistance is difficult due to the highly heterozygous nature andoutcrossing behavior of grapevines, and due to polygenic patterns ofinheritance. Moreover, introduction of a new cultivar may be prohibitedby custom or law. Recent biotechnology developments have made possiblethe introduction of special traits, such as disease resistance, into anestablished cultivar without altering its horticultural characteristics.

Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses,and nematodes can infect grapes, and the resultant diseases can causesubstantial losses in production (Pearson et al., Compendium of GrapeDiseases, American Phytopathological Society Press (1988)). Among these,viral diseases constitute a major hindrance to profitable growing ofgrapevines. About 34 viruses have been isolated and characterized fromgrapevines. The major virus diseases are grouped into: (1) the grapevinedegeneration caused by the fanleaf nepovirus, other Europeannepoviruses, and American nepoviruses, (2) the leafroll complex, and (3)the rugose wood complex (Martelli, ed., Graft Transmissible Diseases ofGrapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy(1993)).

Of the major virus diseases, the grapevine leafroll complex is the mostwidely distributed throughout the world. According to Goheen (“GrapeLeafroll,” in Frazier et al., eds., Virus Diseases of Small Fruits andGrapevines (A Handbook), University of California, Division ofAgricultural Sciences, Berkeley, Calif., USA, pp. 209-212 (1970)(“Goheen (1970)”), grapevine leafroll-like disease was described asearly as the 1850s in German and French literature. However, the viralnature of the disease was first demonstrated by Scheu (Scheu, “DieRollkrankheit des Rebstockes (Leafroll of grapevine),” D. D. Weinbau 14:222-358 (1935) (“Scheu (1935)”)). In 1946, Harmon and Snyder (Harmon etal., “Investigations on the Occurrence, Transmission, Spread and Effectof ‘White’ Fruit Colour in the Emperor Grape,” Proc. Am. Soc. Hort. Sci.74: 190-194 (1946)) determined the viral nature of White Emperor diseasein California. It was later proven by Goheen et al. (Goheen et al.,“Leafroll (White Emperor Disease) of Grapes in California,Phytopathology, 48: 51-54 (1958) (“Goheen (1958)”)) that both leafrolland “White Emperor” diseases were the same, and only the name “leafroll”was retained.

Leafroll is a serious viral disease of grapes and occurs wherever grapesare grown. This wide distribution of the disease has come about throughthe propagation of diseased vines. It affects almost all cultivated androotstock varieties of Vitis. Although the disease is not lethal, itcauses yield losses and reduction of sugar content. Scheu estimated in1936 that 80 percent of all grapevines planted in Germany were infected(Scheu, Mein Winzerbuch, Berlin:Reichsnahrstand-Verlags (1936)). In manyCalifornia wine grape vineyards, the incidence of leafroll (based on asurvey of field symptoms conducted in 1959) agrees with Scheu's initialobservation in German vineyards (Goheen et al., “Studies of GrapeLeafroll in California,” Amer. J. Enol. Vitic., 10: 78-84 (1959)). Thecurrent situation on leafroll disease does not seem to be any better(Goheen, “Diseases Caused by Viruses and Viruslike Agents,” The AmericanPhytopathological Society, St. Paul, Minn.:APS Press, 1: 47-54 (1988)(“Goheen (1988)”). Goheen also estimated that the disease causes anannual loss of about 5-20 percent of the total grape production (Goheen(1970) and Goheen (1988)). The amount of sugar in individual berries ofinfected vines is only about ½ to ⅔ that of berries from noninfectedvines (Goheen (1958)).

Symptoms of leafroll disease vary considerably depending upon thecultivar, environment, and time of the year. On red or dark-coloredfruit varieties, the typical downward rolling and interveinal reddeningof basal, mature leaves is the most prevalent in autumn; but not inspring or early summer. On light-colored fruit varieties however,symptoms are less conspicuous, usually with downward rolling accompaniedby interveinal chlorosis. Moreover, many infected rootstock cultivars donot develop symptoms. In these cases, the disease is usually diagnosedwith a woody indicator indexing assay using Vitis vivifera cv. CarbernetFranc (Goheen (1988)).

Ever since Scheu demonstrated that leafroll was graft transmissible, avirus etiology has been suspected (Scheu (1935)). Several virus particletypes have been isolated from leafroll diseased vines. These includepotyvirus-like (Tanne et al., “Purification and Characterization of aVirus Associated with the Grapevine Leafroll Disease,” Phytopathology67: 442-447 (1977)), isometric virus-like (Castellano et al.,“Virus-like Particles and Ultrastructural Modifications in the Phloem ofLeafroll-affected Grapevines,” Vitis, 22: 23-39 (1983) (“Castellano(1983)”) and Namba et al., “A Small Spherical Virus Associated with theAjinashika Disease of Koshu Grapevine, Ann. Phytopathol. Soc. Japan, 45:70-73 (1979)), and closterovirus-like (Namba, “Grapevine Leafroll Virus,a Possible Member of Closteroviruses, Ann. Phytopathol. Soc. Japan, 45:497-502 (1979)) particles. In recent years, however, long flexuousclosteroviruses ranging from 1,400 to 2,200 nm have been mostconsistently associated with leafroll disease (FIG. 1) (Castellano(1983), Faoro et al., “Association of a Possible Closterovirus withGrapevine Leafroll in Northern Italy,” Riv. Patol. Veg., Ser IV, 17:183-189 (1981), Gugerli et al., “L'enroulement de la vigne: mise enévidence de particules virales et développement d'une méthodeimmuno-enzymatique pour le diagnostic rapide (Grapevine Leafroll:Presence of Virus Particles and Development of an Immuno-enzyme methodfor Diagnosis and Detection),” Rev. Suisse Viticult. Arboricult, Hort.,16: 299-304 (1984) (“Gugerli (1984)”), Hu et al., “Characterization ofClosterovirus-like Particles Associated with Grapevine LeafrollDisease,” J. Phptopathol., 128: 1-14 (1990) (“Hu (1990)”), Milne et al.,“Closterovirus-like Particles of Two Types Associated with DiseasedGrapevines,” Phytopathol. Z., 110: 360-368 (1984), Zee et al.,“Cytopathology of Leafroll-diseased Grapevines and the Purification andSerology of Associated Closterovirus like Particles,” Phytopathology 77:1427-1434 (1987) (“Zee (1987)”), and Zimmermann et al.,“Characterization and Serological Detection of Four Closterovirus-likeParticles Associated with Leafroll Disease on Grapevine,” J.Phytopathol., 130: 205-218 (1990) (“Zimmermann (1990)”)). Theseclosteroviruses are referred to as grapevine leafroll associated viruses(“GLRaV”). At least six serologically distinct types of GLRaV's (GLRaV-1to −6) have been detected from leafroll diseased vines (Table 1) (Bosciaet al., “Nomenclature of Grapevine Leafroll-associated PutativeClosteroviruses, Vitis, 34: 171-175 (1995) (“Boscia (1995)”) and(Martelli, “Leafroll,” pp. 37-44 in Martelli, ed., Graft TransmissibleDiseases of Graipevines. Handbook for Detection and Diagnosis, FAO, RomeItaly, (1993) (“Martelli I”)). The first five of these were confirmed inthe 10th. Meeting of the International Council for the Study of Virusand Virus Diseases of the Grapevine (“ICVG”) (Volos, Greece, 1990).TABLE 1 Particle Coat length protein Mr Type (nm) (X10³) ReferenceGLRaV-1 1,400-2,200 39 Gugerli (1984) GLRaV-2 1,400-1,800 26 Gugerli(1984) Zimmermann (1990) GLRaV-3 1,400-2,200 43 Zee (1987) GLRaV-41,400-2,200 36 Hu (1990) GLRaV-5 1,400-2,200 36 Zimmermann (1990)GLRaV-6 1,400-2,200 36 Gugerli (1993)

Through the use of monoclonal antibodies, however, the original GLRaV IIdescribed in Gugerli (1984) has been shown to be an apparent mixture ofat least two components, IIa and IIb (Gugerli et al., “GrapevineLeafroll Associated Virus II Analyzed by Monoclonal Antibodies,” 11thMeeting of the International Council for the Study of Viruses and VirusDiseases of the Grapevine, Montreux, Switzerland, pp. 23-24 (1993)(“Gugerli (1993)”)). Recent investigation with comparative serologicalassays (Boscia (1995)) demonstrated that the IIb component of cv.Chasselas 8/22 is the same as the GLRaV-2 isolate from France(Zimmermann (1990)) which also include the isolates of grapevine corkybark associated closteroviruses from Italy (GCBaV-BA) (Boscia (1995))and from the United States (GCBaV-NY) (Namba et al., “Purification andProperties of Closterovirus-like Particles Associated with GrapevineCorky Bark Disease,” Phytopathology, 81: 964-970 (1991) (“Namba(1991)”)). The Ia component of cv. Chasselas 8/22 was given theprovisional name of grapevine leafroll associated virus 6 (GLRaV-6).Furthermore, the antiserum to the CA-5 isolate of GLRaV-2 produced byBoscia et al. (Boscia et al., “Characterization of Grape LeafrollAssociated Closterovirus (GLRaV) Serotype II and Comparison with GLRaVSerotype III,” Phytopathology, 80: 117 (1990)) was shown to containantibodies to both GLRaV-2 and GLRaV-1, with a prevalence of the latter(Boscia (1995)).

Virions of GLRaV-2 are flexuous, filamentous particles about 1,400-1,800nm in length (Gugerli et al., “L'enroulement de la Vigne: Mise enEvidence de Particles Virales et Development d'une MethodeImmuno-enzymatique Pour le Diagnostic Rapide (Grapevine Leafroll:Presence of Virus Particles and Development of an Immuno-enzyme Methodfor Diagnosis and Detection),” Rev. Suisse Viticult. Arboricult.Horticult. 16: 299-304 (1984)). A double-stranded RNA (dsRNA) of about15 kb was consistently isolated from GLRaV-2 infected tissues(Goszczynski et al., “Detection of Two Strains of GrapevineLeafroll-Associated Virus 2,” Vitis 35: 133-35 (1996)). The coat proteinof GLRaV-2 is ca 22˜26 kDa (Zimmermann et al., “Characterization andSerological Detection of Four Closterovirus-like Particles Associatedwith Leafroll Disease on Grapevine,” J. Phytopathology 130: 205-18(1990); Gugerli and Ramel, Extended abstracts: “Grapevine LeafrollAssociated Virus II Analyzed by Monoclonal Antibodies,” 11th ICVG atMontreux, Switzerland, Gugerli, ed., Federal Agricultural ResearchStation of Changins, CH-1260 Nyon, Switzerland, p. 23-24 (1993); Bosciaet al., “Nomenclature of Grapevine Leafroll-Associated PutativeClosteroviruses,” Vitis 34: 171-75 (1995)), which is considerablysmaller than other GLRaVs (35-43 kDa) (Zee et al., “Cytopathology ofLeafroll-Diseased Grapevines and the Purification and Serology ofAssociated Closterovirus Like Particles,” Phytopathology 77: 1427-34(1987); Hu et al., “Characterization of Closterovirus-Like ParticlesAssociated with Grapevine Leafroll Disease,” J. of Phytopathology 128:1-14 (1990); Ling et al., “The Coat Protein Gene of Grapevine LeafrollAssociated Closterovirus-3: Cloning, Nucleotide Sequencing andExpression in Transgenic Plants,” Arch. of Virologv 142: 1101-16(1997)). Although GLRaV-2 has been classififed as a member of the genusClosterovirus based on particle morphology and cytopathology (Martelli,Circular of ICTV-Plant Virus Subcommittee Study Group on ClosterolikeViruses” (1996)), its molecular and biochernical properties are not wellcharacterized.

In the closterovirus group, several viruses have recently beensequenced. The partial or complete genome sequences of beet yellowsvirus (BYV) (Agranovsky et al. “Nucleotide Sequence of the 3′-TerminalHalf of Beet Yellows Closterovirus RNA Genome Unique Arrangement ofEight Virus Genes,” J. General Virology 72: 15-24 (1991); Agranovsky etal., “Beet Yellows Closterovirus: Complete Genome Structure andIdentification of a Papain-like Tiol Protease,” Virology 198: 311-24(1994)), beet yellow stunt virus (BYSV) (Karasev et al., “Organizationof the 3′-Terminal Half of Beet Yellow Stunt Virus Genome andImplications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996)), citrus tristeza virus (CTV) (Pappu et al., “NucleotideSequence and Organization of Eight 3′ Open Reading Frames of the CitrusTristeza Closterovirus Genome,” Virology 199: 35-46 (1994); Karasev etal., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,”Virology 208: 511-20 (1995)), lettuce infectious yellows virus (LIYV)(Klaassen et al., “Partial Characterization of the Lettuce InfectiousYellows Virus Genomic RNAs, Identification of the Coat Protein Gene andComparison of its Amino Acid Sequence With Those of Other FilamentousRNA Plant Viruses,” J. General Virology 75: 1525-33 (1994); Klaassen etal., “Genome Structure and Phylogenetic Analysis of Lettuce InfectiousYellows Virus, a Whitefly-Transmitted, Bipartite Closterovirus,”Virology 208: 99-110 (1995)), little cherry virus (LChV) (Keim andJelkmann, “Genome Analysis of the 3′-Terminal Part of the Little CherryDisease Associated dsRNA Reveals a Monopartite Clostero-Like Virus,”Arch. Virology 141: 1437-51 (1996); Jelkmann et al., “Complete GenomeStructure and Phylogenetic Analysis of Little Cherry Virus, aMealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71(1997)), and GLRaV-3 (Ling et al., “Nucleotide Sequence of the 3′Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 GenomeReveals a Typical Monopartite Closterovirus,” J. Gen. Virology 79(5):1289-1301 (1998)) revealed several common features of theclosteroviruses, including the presence of HSP70 chaperone heat shockprotein and a duplicate of the coat protein gene (Agranovsky “Principlesof Molecular Organization, Expression, and Evolution of Closteroviruses:Over the Barriers,” Adv. in Virus Res. 47: 119-218 (1996); Dolja et al.“Molecular Biology and Evolution of Closteroviruses: SophisticatedBuild-up of Large RNA Genomes,” Annual Rev. Photopathology 32: 261-85(1994); Boyko et al.,. “Coat Protein Gene Duplication in a FilamentousRNA Virus of Plants,” Proc. Nat. Acad. Sci. USA 89: 9156-60 (1992)).Characterization of the genome organization of GLRaVs would providemolecular information on the serologically distinct closteroviruses thatcause similar leafroll symptoms in grapevine.

Several shorter closteroviruses (particle length 800 nm long) have alsobeen isolated from grapevines. One of these, called grapevine virus A(“GVA”) has also been found associated, though inconsistently, with theleafroll disease (Agran et al., “Occurrence of Grapevine Virus A (GVA)and Other Closteroviruses in Tunisian Grapevines Affected by LeafrollDisease,” Vitis, 29: 43-48 (1990), Conti, et al., “ClosterovirusAssociated with Leafroll and Stem Pitting in Grapevine,” Phytopathol.Mediterr., 24: 110-113 (1985), and Conti et al., “A Closterovirus from aStem-pitting-diseased Grapevine,” Phytopathology, 70: 394-399 (1980)).The etiology of GVA is not really known; however, it appears to be moreconsistently associated with rugose wood sensu lato (Rosciglione at al.,“Maladies de l'enroulement et du bois strie de la vigne: analysemicroscopique et serologique (Leafroll and Stem Pitting of Grapevine:Microscopical and Serological Analysis),” Rev. Suisse Vitic Arboric.Hortic., 18: 207-211 (1986) (“Rosciglione (1986)”), and Zimmermann(1990)). Moreover, another short closterovirus (800 nm long) namedgrapevine virus B (“GVB”) has been isolated and characterized from corkybark-affected vines (Boscia et al., “Properties of a Filamentous VirusIsolated from Grapevines Affected by Corky Bark,” Arch. Virol., 130:109-120 (1993) and Namba (1991)).

As suggested by Martelli I, leafroll symptoms may be induced by morethan one virus or they may be simply a general plant physiologicalresponse to invasion by an array of phloem-inhabiting viruses. Evidenceaccumulated in the last 15 years strongly favors the idea that grapevineleafroll is induced by one (or a complex) of long closteroviruses(particle length 1,400 to 2,200 nm).

Grapevine leafroll is transmitted primarily by contaminated scions androotstocks. However, under field conditions, several species ofmealybugs have been shown to be the vector of leafroll (Engelbrecht etal., “Transmission of Grapevine Leafroll Disease and AssociatedClosteroviruses by the Vine Mealybug Planococcus-ficus,”Phytophylactica, 22: 341-346 (1990), Rosciglione, et al., “Transmissionof Grapevine Leafroll Disease and an Associated Closterovirus to HealthyGrapevine by the Mealybug Planococcus ficus,” (Abstract),Phytoparasitica, 17: 63-63 (1989), and Tanne, “Evidence for theTransmission by Mealybugs to Healthy Grapevines of a Closter-likeParticle Associated with Grapevine Leafroll Disease,” Phytoparasitica,16: 288 (1988)). Natural spread of leafroll by insect vectors is rapidin various parts of the world. In New Zealand, observations of threevineyards showed that the number of infected vines nearly doubled in asingle year (Jordan et al., “Spread of Grapevine Leafroll and itsAssociated Virus in New Zealand Vineyards,” 11th Meeting of theInternational Council for the Study of Viruses and Virus Diseases of theGrapevine, Montreux, Switzerland, pp. 113-114 (1993)). One vineyardbecame 90% infected 5 years after GLRaV-3 was first observed. Prevalenceof leafroll worldwide may increase as chemical control of mealybugsbecomes more difficult due to the unavailability of effectiveinsecticides.

In view of the serious risk grapevine leafroll virus poses to vineyardsand the absence of an effective treatment of it, the need to preventthis affliction continues to exist. The present invention is directed toovercoming this deficiency in the art.

SUMMARY OF INVENTION

The present invention relates to an isolated protein or polypeptidecorresponding to a protein or polypeptide of a grapevine leafroll virus(type 2). The encoding RNA and DNA molecules, in either isolated form orincorporated in an expression system, a host cell, a transgenic Vitis orcitrus scion or rootstock cultivar, or a transgenic Nicotiana plant orbeet plant are also disclosed.

Another aspect of the present invention relates to a method of impartinggrapevine leafroll virus (type 2) resistance to Vitis scion or rootstockcultivars or Nicotiana plants by transforming them with a DNA moleculeencoding the protein or polypeptide corresponding to a protein orpolypeptide of a grapevine leafroll virus (type 2). Other aspects of thepresent invention relate to a method of imparting beet yellows virusresistance to beet plants and a method of imparting tristeza virusresistance to citrus scion or rootstock cultivars, both by transformingthe plants or cultivars with a DNA molecule encoding the protein orpolypeptide corresponding to a protein or polypeptide of a grapevineleafroll virus (type 2).

The present invention also relates to an antibody or binding portionthereof or probe which recognizes the protein or polypeptide.

Grapevine leafroll virus resistant transgenic variants of the currentcommercial grape cultivars and rootstocks allows for more completecontrol of the virus, while retaining the varietal characteristics ofspecific cultivars. Furthermore, these variants permit control ofGLRaV-2 transmitted either by contaminated scions or rootstocks or by apresently uncharacterized insect vector. With respect to the latter modeof transmission, the present invention circumvents increased restrictionof pesticide use which has made chemical control of insect infestationincreasingly difficult. In this manner, the interests of the environmentand the economics of grape cultivation and wine making are all furtheredby the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a comparison of a double-stranded RNA (dsRNA)profile (FIG. 1A) of GLRaV-2 and its Northern hybridization analysis(FIG. 1B). In FIG. 1A: lane M, lambda Hind III DNA marker; and lane 1,dsRNA pattern in 1% agarose gel stained with ethidium bromide. FIG. 1Bis a northern hybridization of isolated high molecular weight dsRNA ofGLRaV-2 with a probe prepared with ³²P [α-dATP] labeled cDNA insert fromGLRaV-2 specific cDNA clone TC-1. Lane 1, high molecular weight dsRNA ofGLRaV-2. Lane 2, total RNA extracted from healthy grapevine.

FIG. 2 displays the genome organization of GLRaV-2 and its sequencingstrategy. Boxes represent ORFs encoded by deduced amino acid sequencesof GLRaV-2, numbered lines represent nucleotide coordinates, beginningfrom 5′-terminal of RNA in kilobases (kb). The lines below GLRaV-2 RNAgenome represent the cDNA clones used to determine the nucleotidesequences.

FIG. 3A-3D are comparisons between ORF1a/ORF1b of GLRaV-2 and BYV. FIG.3A-3D show the conserved domains of two papain-like proteases (P-PRO),methyltransferase (MT/MTR), helicase (HEL), and RNA-dependent RNApolymerase (RdRP), respectively. Exclamation marks indicate thepredicted catalytic residues of the leader papain-like protease; slashesindicate the predicted cleavage sites. The conserved motifs of the MT,HEL, and RdRP domains are highlighted with overlines marked withrespective letters. The alignment is constructed using the MegAlignprogram in DNASTAR.

FIGS. 4A and 4B are alignments of the nucleotide (FIG. 4A) and deducedamino acid (FIG. 4B) sequences of ORF1a/ORF1b overlapping region ofGLRaV-2, BYV, BYSV, and CTV. Identical nucleotides and amino acids areshown in consensus. GLRaV-2 putative +1 frameshift site (TAGC) and itscorresponding sites of BYV (TAGC) and BYSV (TAGC) and CTV (CGGC) atnucleotide and amino acid sequences are highlighted with underlines.

FIG. 5 is an alignment of the amino acid sequence of HSP70 protein ofGLRaV-2 and BYV. The conserved motifs (A to H) are indicated withoverlines and marked with respective letters. The alignment wasconducted with the MegAlign program of DNASTAR.

FIG. 6A is a comparison of the coat protein (CP) and coat proteinduplicate (CPd) of GLRaV-2 with other closteroviruses. The amino acidsequence of the GLRaV-2 CP and CPd are aligned with the CP and CPd ofBYV, BYSV, and CTV. The conserved amino acid residues are in bold andthe consensus sequences are indicated. Sequence alignment andphylogenetic tree were constructed by Clustal Method in the MegAlignProgram of DNASTAR. FIG. 6B is a tentative phylogenetic tree of the CPand CPd of GLRaV-2 with BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3. Tofacilitate the alignment, only the C-terminal 250 amino acids of CP andCPd of LIYV, LChV, and GLRaV-3 were used. The scale beneath thephylogenetic tree represents the distance between sequences. Unitsindicate the number of substitution events.

FIG. 7 is a comparison of the genome organization of GLRaV-2, BYV, BYSV,CTV, LIYV, LChV, and GLRaV-3. P—PRO, papain-like protease; MT/MTR,methyltransferase; HEL, helicase; RdRP, RNA-dependent RNA polymerase;HSP70, heat shock protein 70; CP, coat protein; CPd, coat proteinduplicate.

FIG. 8 is a tentative phylogenetic tree showing the relationship of RdRPof GLRaV-2 with respect to BYV, BYSV, CTV, and LIYV. The phylogenetictree was constructed using the Clustal method with the MegAlign programin DNASTAR.

FIG. 9 is an alignment of the amino acid sequence of HSP90 protein ofGLRaV-2 with respect to other closteroviruses, BYS, BYSV, and CTV. Themost conserved motifs (I to II) are indicated with the highlighted linesand marked with respective letters.

FIG. 10 is an alignment of the nucleotide sequence of 3′-terminaluntranslated region of GLRaV-2 with respect to the closteroviruses BYV(Agranovsky et al., “Beet Yellows Closterovirus: Complete GenomeStructure and Identification of a Papain-like Thiol Protease,” Virology198: 311-24 (1994), which is hereby incorporated by reference), BYSV(Karasev et al., Organization of the 3′-Terminal Half of Beet YellowStunt Virus Genome and Implications for the Evolution ofClosteroviruses,” Virology 221: 199-207 (1996), which is herebyincorporated by reference), and CTV (Karasev et al., “Complete Sequenceof the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995),which is hereby incorporated by reference). The consensus sequences areshown, and the distance to the 3′-end is indicated. A complementaryregion capable of forming a “hair-pin” structure is underlined.

FIGS. 11A and 11B are genetic maps of the transformation vectorspGA482GG/EPT8CP-GLRaV-2 and pGA482G/EPT8CP-GLRaV-2, respectively. Asshown in FIGS. 11A and 11B, the plant expression cassette(EPT8CP-GLRaV-2), which consists of a double cauliflower mosaic virus(CaMV) 35S-enhancer, a CaMV 35S-promoter, an alfalfa mosaic virus (ALMV)RNA4 5′ leader sequence, a coat protein gene of GLRaV-2 (CP-GLRaV-2),and a CaMV 35S 3′ untranslated region as a terminator, was cloned intothe transformation vector by EcoR I restriction site. The CP of GLRaV-2was cloned into the plant expression vector by Nco I restriction site.

FIG. 12 is a PCR analysis of DNA molecules extracted from the leaves ofputative transgenic plants using both the CP gene of GLRaV-2 and NPT IIgene specific primers. An ethidium bromide-stained gel shows a 720 bpamplified DNA fragment for NPT II gene, and a 653 bp DNA fragment forthe entire coding sequence of the CP gene. Lane 1, φ 174/Hae III DNAMarker; lanes 2-6, transgenic plants from different lines; lane 7, thecp gene of GLRaV-2 of positive control; and lane 8, NPT II gene ofpositive control.

FIG. 13 is a comparison of resistant (right side 3 plants) andsusceptible (left side 3 plants) transgenic Nicotiana benthamianaplants. Plants are shown 48 days after inoculation with GLRaV-2.

FIG. 14 is a northern blot analysis of transgenic Nicotiana benthamianaplants. An aliquot of 10 g of total RNA extracted from putativetransgenic plants was denatured and loaded onto 1% agarose gelcontaining formaldehyde. The separated RNAs were transferred to GeneScreen Plus membrane and hybridized with a ³²P-labeled DNA probecontaining the 3′ one third CP gene sequence. Lanes 1, 3, and 4represent nontransformed control plants without RNA expression. Theremaining lanes represent transgenic plants from different lines: lanes2, 14-17, and 22-27 represent plants with high RNA expression levelwhich are susceptible to GLRaV-2; all other lanes represent plants withundetectable or low RNA expression level which are resistant to GLRaV-2.

DETAILED DESCRIPTION OF TH INVENTION

The present invention relates to isolated DNA molecules encoding for theproteins or polypeptides of a grapevine leafroll virus (type 2). Asubstantial portion of the grapevine leafroll virus (type-2) (“GLRaV-2”)genome has been sequenced. Within the genome are a plurality of openreading frames (“ORFs”) and a 3′ untranscribed region (“UTR”), eachcontaining DNA molecules in accordance with the present invention. TheDNA molecule which constitutes a substantial portion of the GLRaV-2genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 1as follows: TAAACATTGC GAGAGAACCC CATTAGCGTC TCCGGGGTGA ACTTGGGAAGGTCTGCCGCC    60 GCTCAGGTTA TTTATTTCGG CAGTTTCACG CAGCCCTTCG CGTTGTATCCGCGCCAAGAG   120 AGCGCGATCG TAAAAACGCA ACTTCCACCG GTCAGTGTAG TGAAGGTGGAGTGCGTAGCT   180 GCGGAGGTAG CTCCCGACAG GGGCGTGGTC GACAAGAAAC CTACGTCTGTTGGCGTTCCC   240 CCGCAGCGCG GTGTGCTTTC TTTTCCGACG GTGGTTCGGA ACCGCGGCGACGTGATAATC   300 ACAGGGGTGG TGCATGAAGC CCTGAAGAAA ATTAAAGACG GGCTCTTACGCTTCCGCGTA   360 GGCGGTGACA TGCGTTTTTC GAGATTTTTC TCATCGAACT ACGGCTGCAGATTCGTCGCG   420 AGCGTGCGTA CGAACACTAC AGTTTGGCTA AATTGCACGA AAGCGAGTGGTGAGAAATTC   480 TCACTCGCCG CCGCGTGCAC GGCGGATTAC GTGGCGATGC TGCGTTATGTGTGTGGCGGG   540 AAATTTCCAC TCGTCCTCAT GAGTAGAGTT ATTTACCCGG ATGGGCGCTGTTACTTGGCC   600 CATATGAGGT ATTTGTGCGC CTTTTACTGT CGCCCGTTTA GAGAGTCGGATTATGCCCTC   660 GGAATGTGGC CTACGGTGGC GCGTCTCAGG GCATGCGTTG AGAAGAACTTCGGTGTCGAA   720 GCTTGTGGCA TAGCTCTTCG TGGCTATTAC ACCTCTCGCA ATGTTTATCACTGTGATTAT   780 GACTCTGCTT ATGTAAAATA TTTTAGAAAC CTTTCCGGCC GCATTGGCGGTGGTTCGTTG   840 GATCCGACAT CTTTAACCTC CGTAATAACG GTGAAGATTA GCGGTCTTCCAGGTGGTCTT   900 CCTAAAAATA TAGCGTTTGG TGCCTTCCTG TGCGATATAC GTTACGTCGAACCGGTAGAC   960 TCGGGCGGCA TTCAATCGAG CGTTAAGACG AAACGTGAAG ATGCGCACCGAACCGTAGAG  1020 GAACGGGCGG CCGGCGGATC CGTCGAGCAA CCGCGACAAA AGAGGATAGATGAGAAAGGT  1080 TGCGGCAGAG TTCCTAGTGG AGGTTTTTCG CATCTCCTGG TCGGCAACCTTAACGAAGTT  1140 AGGAGGAAGG TAGCTGCCGG ACTTCTACGC TTTCGCGTTG GCGGTGATATGGATTTTCAT  1200 CGCTCGTTCT CCACCCAAGC GGGCCACCGC TTGCTGGTGT GGCGCCGCTCGAGCCGGAGC  1260 GTGTGCCTTG AACTTTACTC ACCATCTAAA AACTTTTTGC GTTACGATGTCTTGGCCTGT  1320 TCTGGAGACT ATGCAGCGAT GTTTTCTTTC GCGGCGGGCG GCCGTTTCCCTTTAGTTTTG  1380 ATGACTAGAA TTAGATACCC GAACGGGTTT TGTTACTTGG CTCACTGCCGGTACGCGTGC  1440 GCGTTTCTCT TAAGGGGTTT TGATCCGAAG CGTTTCGACA TCGGTGCTTTCCCCACCGCG  1500 GCCAAGCTCA GAAACCGTAT GGTTTCGGAG CTTGGTGAAA GAAGTTTAGGTTTGAACTTG  1560 TACGGCGCAT ATACGTCACG CGGCGTCTTT CACTGCGATT ATGACGCTAAGTTTATAAAG  1620 GATTTGCGTC TTATGTCAGC AGTTATAGCT GGAAAGGACG GGGTGGAAGAGGTGGTACCT  1680 TCTGACATAA CTCCTGCCAT GAAGCAGAAA ACGATCGAAG CCGTGTATGATAGATTATAT  1740 GGCGGCACTG ACTCGTTGCT GAAACTGAGC ATCGAGAAAG ACTTAATCGATTTCAAAAAT  1800 GACGTGCAGA GTTTGAAGAA AGATCGGCCG ATTGTCAAAG TGCCCTTTTACATGTCGGAA  1860 GCAACACAGA ATTCGGTGAC GCGTTTCTAC CCTCAGTTCG AACTTAAGTTTTCGCACTCC  1920 TCGCATTCAG ATCATCCCGC CGCCGCCGCT TCTAGACTGC TGGAAAATGAAACGTTAGTG  1980 CGCTTATGTG GTAATAGCGT TTCAGATATT GGAGGTTGTC CTCTTTTCCATTTGCATTCC  2040 AAGACGCAAA GACGGGTTCA CGTATGTAGG CCTGTGTTGG ATGGCAAGGATGCGCAGCGT  2100 CGCGTGGTGC GTGATTTGCA GTATTCCAAC GTGCGTTTGG GAGACGATGATAAAATTTTG  2160 GAAGGGCCAC GCAATATCGA CATTTGCCAC TATCCTCTGG GCGCGTGTGACCACGAAAGT  2220 AGTGCTATGA TGATGGTGCA GGTGTATGAC GCGTCCCTTT ATGAGATATGTGGCGCCATG  2280 ATCAAGAAGA AAAGCCGCAT AACGTACTTA ACCATGGTCA CGCCCGGCGAGTTTCTTGAC  2340 GGACGCGAAT GCGTCTACAT GGAGTCGTTA GACTGTGAGA TTGAAGTTGATGTGCACGCG  2400 GACGTCGTAA TGTACAAATT CGGTAGTTCT TGCTATTCGC ACAAGCTTTCAATCATCAAG  2460 GACATCATGA CCACTCGGTA CTTGACACTA GGTGGTTTTC TATTCAGCGTGGAGATGTAT  2520 GAGGTGCGTA TGGGCGTGAA TTACTTCAAG ATTACGAAGT CCGAAGTATCGCCTAGCATT  2580 AGCTGCACCA AGCTCCTGAG ATACCGAAGA GCTAATAGTG ACGTGGTTAAAGTTAAACTT  2640 CCACGTTTCG ATAAGAAACG TCGCATGTGT CTGCCTGGGT ATGACACCATATACCTAGAT  2700 TCGAAGTTTG TGAGTCGCGT TTTCGATTAT GTCGTGTGTA ATTGCTCTGCCGTGAACTCA  2760 AAAACTTTCG AGTGGGTGTG GAGTTTCATT AAGTCTAGTA AGTCGAGGGTGATTATTAGC  2820 GGTAAAATAA TTCACAAGGA TGTGAATTTG GACCTCAAGT ACGTCGAGAGTTTCGCCGCG  2880 GTTATGTTGG CCTCTGGCGT GCGCAGTAGA CTAGCGTCCG AGTACCTTGCTAAGAACCTT  2940 AGTCATTTTT CGGGAGATTG CTCCTTTATT GAAGCCACGT CTTTCGTGTTGCGTGAGAAA  3000 ATCAGAAACA TGACTCTGAA TTTTAACGAA AGACTTTTAC AGTTAGTGAAGCGCGTTGCC  3060 TTTGCGACCT TGGACGTGAG TTTTCTAGAT TTAGATTCAA CTCTTGAATCAATAACTGAT  3120 TTTGCCGAGT GTAAGGTAGC GATTGAACTC GACGAGTTGG GTTGCTTGAGAGCGGAGGCC  3180 GAGAATGAAA AAATCAGGAA TCTGGCGGGA GATTCGATTG CGGCTAAACTCGCGAGCGAG  3240 ATAGTGGTCG ATATTGACTC TAAGCCTTCA CCGAAGCAGG TGGGTAATTCGTCATCCGAA  3300 AACGCCGATA AGCGGGAAGT TCAGAGGCCC GGTTTGCGTG GTGGTTCTAGAAACGGGGTT  3360 GTTGGGGAGT TCCTTCACTT CGTCGTGGAT TCTGCCTTGC GTCTTTTCAAATACGCGACG  3420 GATCAACAAC GGATCAAGTC TTACGTGCGT TTCTTGGACT CGGCGGTCTCATTCTTGGAT  3480 TACAACTACG ATAATCTATC GTTTATACTG CGAGTGCTTT CGGAAGGTTATTCGTGTATG  3540 TTCGCGTTTT TGGCGAATCG CGGCGACTTA TCTAGTCGTG TCCGTAGCGCGGTGTGTGCT  3600 GTGAAAGAAG TTGCTACCTC ATGCGCGAAC GCGAGCGTTT CTAAAGCCAAGGTTATGATT  3660 ACCTTCGCAG CGGCCGTGTG TGCTATGATG TTTAATAGCT GCGGTTTTTCAGGCGACGGT  3720 CGGGAGTATA AATCGTATAT AGATCGTTAC ACGCAAGTAT TGTTTGACACTATCTTTTTT  3780 GAGGACAGCA GTTACCTACC CATAGAAGTT CTGAGTTCGG CGATATGCGGTGCTATCGTC  3840 ACACTTTTCT CCTCGGGCTC GTCCATAAGT TTAAACGCCT TCTTACTTCAAATTACCAAA  3900 GGATTCTCCC TAGAGGTTGT CGTCCGGAAT GTTGTGCGAG TCACGCATGGTTTGAGCACC  3960 ACAGCGACCG ACGGCGTCAT ACGTGGGGTT TTCTCCCAAA TTGTGTCTCACTTACTTGTT  4020 GGAAATACGG GTAATGTGGC TTACCAGTCA GCTTTCATTG CCGGGGTGGTGCCTCTTTTA  4080 GTTAAAAAGT GTGTGAGCTT AATCTTCATC TTGCGTGAAG ATACTTATTCCGGTTTTATT  4140 AAGCACGGAA TCAGTGAATT CTCTTTCCTT AGTAGTATTC TGAAGTTCTTGAAGGGTAAG  4200 CTTGTGGACG AGTTGAAATC GATTATTCAA GGGGTTTTTG ATTCCAACAAGCACGTGTTT  4260 AAAGAAGCTA CTCAGGAAGC GATTCGTACG ACGGTCATGC AAGTGCCTGTCGCTGTAGTG  4320 GATGCCCTTA AGAGCGCCGC GGGAAAAATT TATAACAATT TTACTAGTCGACGTACCTTT  4380 GGTAAGGATG AAGGCTCCTC TAGCGACGGC GCATGTGAAG AGTATTTCTCATGCGACGAA  4440 GGTGAAGGTC CGGGTCTGAA AGGGGGTTCC AGCTATGGCT TCTCAATTTTAGCGTTCTTT  4500 TCACGCATTA TGTGGGGAGC TCGTCGGCTT ATTGTTAAGG TGAAGCATGAGTGTTTTGGG  4560 AAACTTTTTG AATTTCTATC GCTCAAGCTT CACGAATTCA GGACTCGCGTTTTTGGGAAG  4620 AATAGAACGG ACGTGGGAGT TTACGATTTT TTGCCCACGG GCATCGTGGAAACGCTCTCA  4680 TCGATAGAAG AGTGCGACCA AATTGAAGAA CTTCTCGGCG ACGACCTGAAAGGTGACAAG  4740 GATGCTTCGT TGACCGATAT GAATTACTTT GAGTTCTCAG AAGACTTCTTAGCCTCTATC  4800 GAGGAGCCGC CTTTCGCTGG ATTGCGAGGA GGTAGCAAGA ACATCGCGATTTTGGCGATT  4860 TTGGAATACG CGCATAATTT GTTTCGCATT GTCGCAAGCA AGTGTTCGAAACGACCTTTA  4920 TTTCTTGCTT TCGCCGAACT CTCAAGCGCC CTTATCGAGA AATTTAAGGAGGTTTTCCCT  4980 CGTAAGAGCC AGCTCGTCGC TATCGTGCGC GAGTATACTC AGAGATTCCTCCGAAGTCGC  5040 ATGCGTGCGT TGGGTTTGAA TAACGAGTTC GTGGTAAAAT CTTTCGCCGATTTGCTACCC  5100 GCATTAATGA AGCGGAAGGT TTCAGGTTCG TTCTTAGCTA GTGTTTATCGCCCACTTAGA  5160 GGTTTCTCAT ATATGTGTGT TTCAGCGGAG CGACGTGAAA AGTTTTTTGCTCTCGTGTGT  5220 TTAATCGGGT TAAGTCTCCC TTTCTTCGTG CGCATCGTAG GAGCGAAAGCGTGCGAAGAA  5280 CTCGTGTCCT CAGCGCGTCG CTTTTATGAG CGTATTAAAA TTTTTCTAAGGCAGAAGTAT  5340 GTCTCTCTTT CTAATTTCTT TTGTCACTTG TTTAGCTCTG ACGTTGATGACAGTTCCGCA  5400 TCTGCAGGGT TGAAAGGTGG TGCGTCGCGA ATGACGCTCT TCCACCTTCTGGTTCGCCTT  5460 GCTAGTGCCC TCCTATCGTT AGGGTGGGAA GGGTTAAAGC TACTCTTATCGCACCACAAC  5520 TTGTTATTTT TGTGTTTTGC ATTGGTTGAC GATGTGAACG TCCTTATCAAAGTTCTTGGG  5580 GGTCTTTCTT TCTTTGTGCA ACCAATCTTT TCCTTGTTTG CGGCGATGCTTCTACAACCG  5640 GACAGGTTTG TGGAGTATTC CGAGAAACTT GTTACAGCGT TTGAATTTTTCTTAAAATGT  5700 TCGCCTCGCG CGCCTGCACT ACTCAAAGGG TTTTTTGAGT GCGTGGCGAACAGCACTGTG  5760 TCAAAAACCG TTCGAAGACT TCTTCGCTGT TTCGTGAAGA TGCTCAAACTTCGAAAAGGG  5820 CGAGGGTTGC GTGCGGATGG TAGGGGTCTC CATCGGCAGA AAGCCGTACCCGTCATACCT  5880 TCTAATCGGG TCGTGACCGA CGGGGTTGAA AGACTTTCGG TAAAGATGCAAGGAGTTGAA  5940 GCGTTGCGTA CCGAATTGAG AATCTTAGAA GATTTAGATT CTGCCGTGATCGAAAAACTC  6000 AATAGACGCA GAAATCGTGA CACTAATGAC GACGAATTTA CGCGCCCTGCTCATGAGCAG  6060 ATGCAAGAAG TCACCACTTT CTGTTCGAAA GCCAACTCTG CTGGTTTGGCCCTGGAAAGG  6120 GCAGTGCTTG TGGAAGACGC TATAAAGTCG GAGAAACTTT CTAAGACGGTTAATGAGATG  6180 GTGAGGAAAG GGAGTACCAC CAGCGAAGAA GTGGCCGTCG CTTTGTCGGACGATGAAGCC  6240 GTGGAAGAAA TCTCTGTTGC TGACGAGCGA GACGATTCGC CTAAGACAGTCAGGATAAGC  6300 GAATACCTAA ATAGGTTAAA CTCAAGCTTC GAATTCCCGA AGCCTATTGTTGTGGACGAC  6360 AACAAGGATA CCGGGGGTCT AACGAACGCC GTGAGGGAGT TTTATTATATGCAAGAACTT  6420 GCTCTTTTCG AAATCCACAG CAAACTGTGC ACCTACTACG ATCAACTGCGCATAGTCAAC  6480 TTCGATCGTT CCGTAGCACC ATGCAGCGAA GATGCTCAGC TGTACGTACGGAAGAACGGC  6540 TCAACGATAG TGCAGGGTAA AGAGGTACGT TTGCACATTA AGGATTTCCACGATCACGAT  6600 TTCCTGTTTG ACGGAAAAAT TTCTATTAAC AAGCGGCGGC GAGGCGGAAATGTTTTATAT  6660 CACGACAACC TCGCGTTCTT GGCGAGTAAT TTGTTCTTAG CCGGCTACCCCTTTTCAAGG  6720 AGCTTCGTCT TCACGAATTC GTCGGTCGAT ATTCTCCTCT ACGAAGCTCCACCCGGAGGT  6780 GGTAAGACGA CGACGCTGAT TGACTCGTTC TTGAAGGTCT TCAAGAAAGGTGAGGTTTCC  6840 ACCATGATCT TAACCGCCAA CAAAAGTTCG CAGGTTGAGA TCCTAAAGAAAGTGGAGAAG  6900 GAAGTGTCTA ACATTGAATG CCAGAAACGT AAAGACAAAA GATCTCCGAAAAAGAGCATT  6960 TACACCATCG ACGCTTATTT AATGCATCAC CGTGGTTGTG ATGCAGACGTTCTTTTCATC  7020 GATGAGTGTT TCATGGTTCA TGCGGGTAGC GTACTAGCTT GCATTGAGTTCACGAGGTGT  7080 CATAAAGTAA TGATCTTCGG GGATAGCCGG CAGATTCACT ACATTGAAAGGAACGAATTG  7140 GACAAGTGTT TGTATGGGGA TCTCGACAGG TTCGTGGACC TGCAGTGTCGGGTTTATGGT  7200 AATATTTCGT ACCGTTGTCC ATGGGATGTG TGCGCTTGGT TAAGCACAGTGTATGGCAAC  7260 CTAATCGCCA CCGTGAAGGG TGAAAGCGAA GGTAAGAGCA GCATGCGCATTAACGAAATT  7320 AATTCAGTCG ACGATTTAGT CCCCGACGTG GGTTCCACGT TTCTGTGTATGCTTCAGTCG  7380 GAGAAGTTGG AAATCAGCAA GCACTTTATT CGCAAGGGTT TGACTAAACTTAACGTTCTA  7440 ACGGTGCATG AGGCGCAAGG TGAGACGTAT GCGCGTGTGA ACCTTGTGCGACTTAAGTTT  7500 CAGGAGGATG AACCCTTTAA ATCTATCAGG CACATAACCG TCGCTCTTTCTCGTCACACC  7560 GACAGCTTAA CTTATAACGT CTTAGCTGCT CGTCGAGGTG ACGCCACTTGCGATGCCATC  7620 CAGAAGGCTG CGGAATTGGT GAACAAGTTT CGCGTTTTTC CTACATCTTTTGGTGGTAGT  7680 GTTATCAATC TCAACGTGAA GAAGGACGTG GAAGATAACA GTAGGTGCAAGGCTTCGTCG  7740 GCACCATTGA GCGTAATCAA CGACTTTTTG AACGAAGTTA ATCCCGGTACTGCGGTGATT  7800 GATTTTGGTG ATTTGTCCGC GGACTTCAGT ACTGGGCCTT TTGAGTGCGGTGCCAGCGGT  7860 ATTGTGGTGC GGGACAACAT CTCCTCCAGC AACATCACTG ATCACGATAAGCAGCGTGTT  7920 TAGCGTAGTT CGGTCGCAGG CGATTCCGCG TAGAAAACCT TCTCTACAAGAAAATTTGTA  7980 TTCGTTTGAA GCGCGGAATT ATAACTTCTC GACTTGCGAC CGTAACACATCTGCTTCAAT  8040 GTTCGGAGAG GCTATGGCGA TGAACTGTCT TCGTCGTTGC TTCGACCTAGATGCCTTTTC  8100 GTCCCTGCGT GATGATGTGA TTAGTATCAC ACGTTCAGGC ATCGAACAATGGCTGGAGAA  8160 ACGTACTCCT AGTCAGATTA AAGCATTAAT GAAGGATGTT GAATCGCCTTTGGAAATTGA  8220 CGATGAAATT TGTCGTTTTA AGTTGATGGT GAAGCGTGAC GCTAAGGTGAAGTTAGACTC  8280 TTCTTGTTTA ACTAAACACA GCGCCGCTCA AAATATCATG TTTCATCGCAAGAGCATTAA  8340 TGCTATCTTC TCTCCTATCT TTAATGAGGT GAAAAACCGA ATAATGTGCTGTCTTAAGCC  8400 TAACATAAAG TTTTTTACGG AGATGACTAA CAGGGATTTT GCTTCTGTTGTCAGCAACAT  8460 GCTTGGTGAC GACGATGTGT ACCATATAGG TGAAGTTGAT TTCTCAAAGTACGACAAGTC  8520 TCAAGATGCT TTCGTGAAGG CTTTTGAAGA AGTAATGTAT AAGGAACTCGGTGTTGATGA  8580 AGAGTTGCTG GCTATCTGGA TGTGCGGCGA GCGGTTATCG ATAGCTAACACTCTCGATGG  8640 TCAGTTGTCC TTCACGATCG AGAATCAAAG GAAGTCGGGA GCTTCGAACACTTGGATTGG  8700 TAACTCTCTC GTCACTTTGG GTATTTTAAG TCTTTACTAC GACGTTAGAAATTTCGAGGC  8760 GTTGTACATC TCGGGCGATG ATTCTTTAAT TTTTTCTCGC AGCGAGATTTCGAATTATGC  8820 CGACGACATA TGCACTGACA TGGGTTTTGA GACAAAATTT ATGTCCCCAAGTGTCCCGTA  8880 CTTTTGTTCT AAATTTGTTG TTATGTGTGG TCATAAGACG TTTTTTGTTCCCGACCCGTA  8940 CAAGCTTTTT GTCAAGTTGG GAGCAGTCAA AGAGGATGTT TCAATGGATTTCCTTTTCGA  9000 GACTTTTACC TCCTTTAAAG ACTTAACCTC CGATTTTAAC GACGAGCGCTTAATTCAAAA  9060 GCTCGCTGAA CTTGTGGCTT TAAAATATGA GGTTCAAACC GGCAACACCACCTTGGCGTT  9120 AAGTGTGATA CATTGTTTGC GTTCGAATTT CCTCTCGTTT AGCAAGTTATATCCTCGCGT  9180 GAAGGGATGG CAGGTTTTTT ACACGTCGGT TAAGAAAGCG CTTCTCAAGAGTGGGTGTTC  9240 TCTCTTCGAC AGTTTCATGA CCCCTTTTGG TCAGGCTGTC ATGGTTTGGGATGATGAGTA  9300 GCGCTAACTT GTGCGCAGTT TCTTTGTTCG TGACATACAC CTTGTGTGTCACCGTGCGTT  9360 TATAATGAAT CAGGTTTTGC AGTTTGAATG TTTGTTTCTG CTGAATCTCGCGGTTTTTGC  9420 TGTGACTTTC ATTTTCATTC TTCTGGTCTT CCGCGTGATT AAGTCTTTTCGCCAGAAGGG  9480 TCACGAAGCA CCTGTTCCCG TTGTTCGTGG CGGGGGTTTT TCAACCGTAGTGTAGTCAAA  9540 AGACGCGCAT ATGGTAGTTT TCGGTTTGGA CTTTGGCACC ACATTCTCTACGGTGTGTGT  9600 GTACAAGGAT GGACGAGTTT TTTCATTCAA GCAGAATAAT TCGGCGTACATCCCCACTTA  9660 CCTCTATCTC TTCTCCGATT CTAACCACAT GACTTTTGGT TACGAGGCCGAATCACTGAT  9720 GAGTAATCTG AAAGTTAAAG GTTCGTTTTA TAGAGATTTA AAACGTTGGGTGGGTTGCGA  9780 TTCGAGTAAC CTCGACGCGT ACCTTGACCG TTTAAAACCT CATTACTCGGTCCGCTTGGT  9840 TAAGATCGGC TCTGGCTTGA ACGAAACTGT TTCAATTGGA AACTTCGGGGGCACTGTTAA  9900 GTCTGAGGCT CATCTGCCAG GGTTGATAGC TCTCTTTATT AAGGCTGTCATTAGTTGCGC  9960 GGAGGGCGCG TTTGCGTGCA CTTGCACCGG GGTTATTTGT TCAGTACCTGCCAATTATGA 10020 TAGCGTTCAA AGGAATTTCA CTGATCAGTG TGTTTCACTC AGCGGTTATCAGTGCGTATA 10080 TATGATCAAT GAACCTTCAG CGGCTGCGCT ATCTGCGTGT AATTCGATTGGAAAGAAGTC 10140 CGCAAATTTG GCTGTTTACG ATTTCGGTGG TGGGACCTTC GACGTGTCTATCATTTCATA 10200 CCGCAACAAT ACTTTTGTTG TGCGAGCTTC TGGAGGCGAT CTAAATCTCGGTGGAAGGGA 10260 TGTTGATCGT GCGTTTCTCA CGCACCTCTT CTCTTTAACA TCGCTGGAACCTGACCTCAC 10320 TTTGGATATC TCGAATCTGA AAGAATCTTT ATCAAAAACG GACGCAGAGATAGTTTACAC 10380 TTTGAGAGGT GTCGATGGAA GAAAAGAAGA CGTTAGAGTA AACAAAAACATTCTTACGTC 10440 GGTGATGCTC CCCTACGTGA ACAGAACGCT TAAGATATTA GAGTCAACCTTAAAATCGTA 10500 TGCTAAGAGT ATGAATGAGA GTGCGCGAGT TAAGTGCGAT TTAGTGCTGATAGGAGGATC 10560 TTCATATCTT CCTGGCCTGG CAGACGTACT AACGAAGCAT CAGAGCGTTGATCGTATCTT 10620 AAGAGTTTCG GATCCTCGGG CTGCCGTGGC CGTCGGTTGC GCATTATATTCTTCATGCCT 10680 CTCAGGATCT GGGGGGTTGC TACTGATCGA CTGTGCAGCT CACACTGTCGCTATAGCGGA 10740 CAGAAGTTGT CATCAAATCA TTTGCGCTCC AGCGGGGGCA CCGATCCCCTTTTCAGGAAG 10800 CATGCCTTTG TACTTAGCCA GGGTCAACAA GAACTCGCAG CGTGAAGTCGCCGTGTTTGA 10860 AGGGGAGTAC GTTAAGTGCC CTAAGAACAG AAAGATCTGT GGAGCAAATATAAGATTTTT 10920 TGATATAGGA GTGACGGGTG ATTCGTACGC AGCCGTTACC TTCTATATGGATTTCTCCAT 10980 TTCAAGCGTA GGAGCCGTTT CATTCGTGGT GAGAGGTCCT GAGGGTAAGCAAGTGTCACT 11040 CACTGGAACT CCAGCGTATA ACTTTTCGTC TGTGGCTCTC GGATCACGCAGTGTCCGAGA 11100 ATTGCATATT AGTTTAAATA ATAAAGTTTT TCTCGGTTTG CTTCTACATAGAAAGGCGGA 11160 TCGACGAATA CTTTTCACTA AGGATGAAGC GATTCGATAC GCCGATTCAATTGATATCGC 11220 GGATGTGCTA AAGGAATATA AAAGTTACGC GGCCAGTGCC TTACCACCAGACGAGGATGT 11280 CGAATTACTC CTGGGAAAGT CTGTTCAAAA AGTTTTACGG GGAAGCAGACTGGAAGAAAT 11340 ACCTCTCTAG GAGCATAGCA GCACACTCAA GTGAAATTAA AACTCTACCAGACATTCGAT 11400 TGTACGGCGG TAGGGTTGTA AAGAAGTCCG AATTCGAATC AGCACTTCCTAATTCTTTTG 11460 AACAGGAATT AGGACTGTTC ATACTGAGCG AAGGGGAAGT GGGATGGAGCAAATTATGCG 11520 GAATAACGGT GGAAGAAGCA GCATACGATC TTACGAATCC CAAGGCTTATAAATTCACTG 11580 CCGAGAGATG TAGCCGGGAT GTAAAAGGTG AAGGACAAAA ATACTCTATGGAAGACGTGA 11640 TGAATTTCAT GCGTTTATCA AATCTGGATG TTAACGACAA GATGCTGACGGAACAGTGTT 11700 GGTCGCTGTC CAATTCATGC GGTGAATTGA TCAACCCAGA CGACAAAGGGCGATTCGTGG 11760 CTCTCACCTT TAAGGACAGA GACACAGCTG ATGACACGGG TGCCGCCAACGTGGAATGTC 11820 GCGTGGGCGA CTATCTAGTT TACGCTATGT CCCTGTTTGA GCAGAGGACCCAAAAATCGC 11880 AGTCTGGCAA CATCTCTCTG TACGAAAAGT ACTGTGAATA CATCAGGACCTACTTAGGGA 11940 GTACAGACCT GTTCTTCACA GCGCCGGACA GGATTCCGTT ACTTACGGGCATCCTATACG 12000 ATTTTTGTAA GGAATACAAC GTTTTCTACT CGTCATATAA GAGAAACGTCGATAATTTCA 12060 GATTCTTCTT GGCGAATTAT ATGCCTTTGA TATCTGACGT CTTTGTCTTCCAGTGGGTAA 12120 AACCCGCGCC GGATGTTCGG CTGCTTTTTG AGTTAAGTGC AGCGGAACTAACGCTGGAGG 12180 TTCCCACACT GAGTTTGATA GATTCTCAAG TTGTGGTAGG TCATATCTTAAGATACGTAG 12240 AATCCTACAC ATCAGATCCA GCCATCGACG CGTTAGAAGA CAAACTGGAAGCGATACTGA 12300 AAAGTAGCAA TCCCCGTCTA TCGACAGCGC AACTATGGGT TGGTTTCTTTTGTTACTATG 12360 GTGAGTTTCG TACGGCTCAA AGTAGAGTAG TGCAAAGACC AGGCGTATACAAAACACCTG 12420 ACTCAGTGGG TGGATTTGAA ATAAACATGA AAGATGTTGA GAAATTCTTCGATAAACTTC 12480 AGAGAGAATT GCCTAATGTA TCTTTGCGGC GTCAGTTTAA CGGAGCTAGAGCGCATGAGG 12540 CTTTCAAAAT ATTTAAAAAC GGAAATATAA GTTTCAGACC TATATCGCGTTTAAACGTGC 12600 CTAGAGAGTT CTGGTATCTG AACATAGACT ACTTCAGGCA CGCGAATAGGTCCGGGTTAA 12660 CCGAAGAAGA AATACTCATC CTAAACAACA TAAGCGTTGA TGTTAGGAAGTTATGCGCTG 12720 AGAGAGCGTG CAATACCCTA CCTAGCGCGA AGCGCTTTAG TAAAAATCATAAGAGTAATA 12780 TACAATGATC ACGCGAAGAG CGGAGGATTA AAGACCCATT GGTAGTCCTGAAAGACACTT 12840 TATATGAGTT CCAACACAAG CGTGCCGGTT GGGGGTCTCG AAGCACTCGAGACCTCGGGA 12900 GTCGTGCTGA CCACGCGAAA GGAAGCGGTT GATAAGTTTT TTAATGAACTAAAAAACGAA 12960 AATTACTCAT CAGTTGACAG CAGCCGATTA AGCGATTCGG AAGTAAAAGAAGTGTTAGAG 13020 AAAAGTAAAG AAAGTTTCAA AAGCGAACTG GCCTCCACTG ACGAGCACTTCGTCTACCAC 13080 ATTATATTTT TCTTAATCCG ATGTGCTAAG ATATCGACAA GTGAAAAGGTGAAGTACGTT 13140 GGTAGTCATA CGTACGTGGT CGACGGAAAA ACGTACACCG TTCTTGACGCTTGGGTATTC 13200 AACATGATGA AAAGTCTCAC GAAGAAGTAC AAACGAGTGA ATGGTCTGCGTGCGTTCTGT 13260 TGCGCGTGCG AAGATCTATA TCTAACCGTC GCACCAATAA TGTCAGAACGCTTTAAGACT 13320 AAAGCCGTAG GGATGAAAGG TTTGCCTGTT GGAAAGGAAT ACTTAGGCGCCGACTTTCTT 13380 TCGGGAACTA GCAAACTGAT GAGCGATCAC GACAGGGCGG TCTCCATCGTTGCAGCGAAA 13440 AACGCTGTCG ATCGTAGCGC TTTCACGGGT GGGGAGAGAA AGATAGTTAGTTTGTATGAT 13500 CTAGGGAGGT ACTAAGCACG GTGTGCTATA GTGCGTGCTA TAATAATAAACACTAGTGCT 13560 TAAGTCGGGC AGAAGAAAAC GCTATGGAGT TGATGTCCGA CAGCAACCTTAGCAACCTGG 13620 TGATAACCGA CGCCTCTAGT CTAAATGGTG TCGACAAGAA GCTTTTATCTGCTGAAGTTG 13680 AAAAAATGTT GGTGCAGAAA GGGGCTCCTA ACGAGGGTAT AGAAGTGGTGTTCGGTCTAC 13740 TCCTTTACGC ACTCGCGGCA AGAACCACGT CTCCTAAGGT TCAGCGCGCAGATTCAGACG 13800 TTATATTTTC AAATAGTTTC GGAGAGAGGA ATGTGGTAGT AACAGAGGGTGACCTTAAGA 13860 AGGTACTCGA CGGGTGTGCG CCTCTCACTA GGTTCACTAA TAAACTTAGAACGTTCGGTC 13920 GTACTTTCAC TGAGGCTTAC GTTGACTTTT GTATCGCGTA TAAGCACAAATTACCCCAAC 13980 TCAACGCCGC GGCGGAATTG GGGATTCCAG CTGAAGATTC GTACTTAGCTGCAGATTTTC 14040 TGGGTACTTG CCCGAAGCTC TCTGAATTAC AGCAAAGTAG GAAGATGTTCGCGAGTATGT 14100 ACGCTCTAAA AACTGAAGGT GGAGTGGTAA ATACACCAGT GAGCAATCTGCGTCAGCTAG 14160 GTAGAAGGGA AGTTATGTAA TGGAAGATTA CGAAGAAAAA TCCGAATCGCTCATACTGCT 14220 ACGCACGAAT CTGAACACTA TGCTTTTAGT GGTCAAGTCC GATGCTAGTGTAGAGCTGCC 14280 TAAACTACTA ATTTGCGGTT ACTTACGAGT GTCAGGACGT GGGGAGGTGACGTGTTGCAA 14340 CCGTGAGGAA TTAACAAGAG ATTTTGAGGG CAATCATCAT ACGGTGATCCGTTCTAGAAT 14400 CATACAATAT GACAGCGAGT CTGCTTTTGA GGAATTCAAC AACTCTGATTGCGTAGTGAA 14460 GTTTTTCCTA GAGACTGGTA GTGTCTTTTG GTTTTTCCTT CGAAGTGAAACCAAAGGTAG 14520 AGCGGTGCGA CATTTGCGCA CCTTCTTCGA AGCTAACAAT TTCTTCTTTGGATCGCATTG 14580 CGGTACCATG GAGTATTGTT TGAAGCAGGT ACTAACTGAA ACTGAATCTATAATCGATTC 14640 TTTTTGCGAA GAAAGAAATC GTTAAGATGA GGGTTATAGT GTCTCCTTATGAAGCTGAAG 14700 ACATTCTGAA AAGATCGACT GACATGTTAC GAAACATAGA CAGTGGGGTCTTGAGCACTA 14760 AAGAATGTAT CAAGGCATTC TCGACGATAA CGCGAGACCT ACATTGTGCGAAGGCTTCCT 14820 ACCAGTGGGG TGTTGACACT GGGTTATATC AGCGTAATTG CGCTGAAAAACGTTTAATTG 14880 ACACGGTGGA GTCAAACATA CGGTTGGCTC AACCTCTCGT GCGTGAAAAAGTGGCGGTTC 14940 ATTTTTGTAA GGATGAACCA AAAGAGCTAG TAGCATTCAT CACGCGAAAGTACGTGGAAC 15000 TCACGGGCGT GGGAGTGAGA GAAGCGGTGA AGAGGGAAAT GCGCTCTCTTACCAAAACAG 15060 TTTTAAATAA AATGTCTTTG GAAATGGCGT TTTACATGTC ACCACGAGCGTGGAAAAACG 15120 CTGAATGGTT AGAACTAAAA TTTTCACCTG TGAAAATCTT TAGAGATCTGCTATTAGACG 15180 TGGAAACGCT CAACGAATTG TGCGCCGAAG ATGATGTTCA CGTCGACAAAGTAAATGAGA 15240 ATGGGGACGA AAATCACGAC CTCGAACTCC AAGACGAATG TTAAACATTGGTTAAGTTTA 15300 ACGAAAATGA TTAGTAAATA ATAAATCGAA CGTGGGTGTA TCTACCTGACGTATCAACTT 15360 AAGCTGTTAC TGAGTAATTA AACCAACAAG TGTTGGTGTA ATGTGTATGTTGATGTAGAG 15420 AAAAATCCGT TTGTAGAACG GTGTTTTTCT CTTCTTTATT TTTAAAAAAAAAATAAAAAA 15480 AAAAAAAAAA AAGCGGCCGC 15500

Another DNA molecule of the present invention (GLRaV-2 ORF1a) includesnucleotides 4-7923 of SEQ. ID. No. 1 and is believed to code for alarge, grapevine leafroll virus polyprotein containing the conserveddomains characteristic of two papain-like proteases, amethyltransferase, and a helicase. This DNA molecule comprises thenucleotide sequence corresponding to SEQ. ID. No. 2 as follows:ACATTGCGAG AGAACCCCAT TAGCGTCTCC GGGGTGAACT TGGGAAGGTC TGCCGCCGCT   60CAGGTTATTT ATTTCGGCAG TTTCACGCAG CCCTTCGCGT TGTATCCGCG CCAAGAGAGC  120GCGATCGTAA AAACGCAACT TCCACCGGTC AGTGTAGTGA AGGTGGAGTG CGTAGCTGCG  180GAGGTAGCTC CCGACAGGGG CGTGGTCGAC AAGAAACCTA CGTCTGTTGG CGTTCCCCCG  240CAGCGCGGTG TGCTTTCTTT TCCGACGGTG GTTCGGAACC GCGGCGACGT GATAATCACA  300GGGGTGGTGC ATGAAGCCCT GAAGAAAATT AAAGACGGGC TCTTACGCTT CCGCGTAGGC  360GGTGACATGC GTTTTTCGAG ATTTTTCTCA TCGAACTACG GCTGCAGATT CGTCGCGAGC  420GTGCGTACGA ACACTACAGT TTGGCTAAAT TGCACGAAAG CGAGTGGTGA GAAATTCTCA  480CTCGCCGCCG CGTGCACGGC GGATTACGTG GCGATGCTGC GTTATGTGTG TGGCGGGAAA  540TTTCCACTCG TCCTCATGAG TAGAGTTATT TACCCGGATG GGCGCTGTTA CTTGGCCCAT  600ATGAGGTATT TGTGCGCCTT TTACTGTCGC CCGTTTAGAG AGTCGGATTA TGCCCTCGGA  660ATGTGGCCTA CGGTGGCGCG TCTCAGGGCA TGCGTTGAGA AGAACTTCGG TGTCGAAGCT  720TGTGGCATAG CTCTTCGTGG CTATTACACC TCTCGCAATG TTTATCACTG TGATTATGAC  780TCTGCTTATG TAAAATATTT TAGAAACCTT TCCGGCCGCA TTGGCGGTGG TTCGTTCGAT  840CCGACATCTT TAACCTCCGT AATAACGGTG AAGATTAGCG GTCTTCCAGG TGGTCTTCCT  900AAAAATATAG CGTTTGGTGC CTTCCTGTGC GATATACGTT ACGTCGAACC GGTAGACTCG  960GGCGGCATTC AATCGAGCGT TAAGACGAAA CGTGAAGATG CGCACCGAAC CGTAGAGGAA 1020CGGGCGGCCG GCGGATCCGT CGAGCAACCG CGACAAAAGA GGATAGATGA GAAAGGTTGC 1080GGCAGAGTTC CTAGTGGAGG TTTTTCGCAT CTCCTGGTCG GCAACCTTAA CGAAGTTAGG 1140AGGAAGGTAG CTGCCGGACT TCTACGCTTT CGCGTTGGCG GTGATATGGA TTTTCATCGC 1200TCGTTCTCCA CCCAAGCGGG CCACCGCTTG CTGGTGTGGC GCCGCTCGAG CCGGAGCGTG 1260TGCCTTGAAC TTTACTCACC ATCTAAAAAC TTTTTGCGTT ACGATGTCTT GCCCTGTTCT 1320GGAGACTATG CAGCGATGTT TTCTTTCGCG GCGGGCGGCC GTTTCCCTTT AGTTTTGATG 1380ACTAGAATTA GATACCCGAA CGGGTTTTGT TACTTGGCTC ACTGCCGGTA CGCGTGCGCG 1440TTTCTCTTAA GGGGTTTTGA TCCGAAGCGT TTCGACATCG GTGCTTTCCC CACCGCGGCC 1500AAGCTCAGAA ACCGTATGGT TTCGGAGCTT GGTGAAAGAA GTTTAGGTTT GAACTTGTAC 1560GGCGCATATA CGTCACGCGG CGTCTTTCAC TGCGATTATG ACGCTAAGTT TATAAAGGAT 1620TTGCGTCTTA TGTCAGCAGT TATAGCTGGA AAGGACGGGG TGGAAGAGGT GGTACCTTCT 1680GACATAACTC CTGCCATGAA GCAGAAAACG ATCGAAGCCG TGTATGATAG ATTATATGGC 1740GGCACTGACT CGTTGCTGAA ACTGAGCATC GAGAAAGACT TAATCGATTT CAAAAATGAC 1800GTGCAGAGTT TGAAGAAAGA TCGGCCGATT GTCAAAGTGC CCTTTTACAT GTCGGAAGCA 1860ACACAGAATT CGCTGACGCG TTTCTACCCT CAGTTCGAAC TTAAGTTTTC GCACTCCTCG 1920CATTCAGATC ATCCCGCCGC CGCCGCTTCT AGACTGCTGG AAAATGAAAC GTTAGTGCGC 1980TTATGTGGTA ATAGCGTTTC AGATATTGGA GGTTGTCCTC TTTTCCATTT GCATTCCAAG 2040ACGCAAAGAC GGGTTCACGT ATGTAGGCCT GTGTTGGATG GCAAGGATGC GCAGCGTCGC 2100GTGGTGCGTG ATTTGCAGTA TTCCAACGTG CGTTTGGGAG ACGATGATAA AATTTTGGAA 2160GGGCCACGCA ATATCGACAT TTGCCACTAT CCTCTGGGCG CGTGTGACCA CGAAAGTAGT 2220GCTATGATGA TGGTGCAGGT GTATGACGCG TCCCTTTATG AGATATGTGG CGCCATGATC 2280AAGAAGAAAA GCCGCATAAC GTACTTAACC ATGGTCACGC CCGGCGAGTT TCTTGACGGA 2340CGCGAATGCG TCTACATGGA GTCGTTAGAC TGTGAGATTG AAGTTGATGT GCACGCGGAC 2400GTCGTAATGT ACAAATTCGG TAGTTCTTGC TATTCGCACA AGCTTTCAAT CATCAAGGAC 2460ATCATGACCA CTCCGTACTT GACACTAGGT GGTTTTCTAT TCAGCGTGGA GATGTATGAG 2520GTGCGTATGG GCGTGAATTA CTTCAAGATT ACGAAGTCCG AAGTATCGCC TAGCATTAGC 2580TGCACCAAGC TCCTGAGATA CCGAAGAGCT AATAGTGACG TGGTTAAAGT TAAACTTCCA 2640CGTTTCGATA AGAAACGTCG CATGTGTCTG CCTGGGTATG ACACCATATA CCTAGATTCG 2700AAGTTTGTGA GTCGCGTTTT CGATTATGTC GTGTGTAATT GCTCTGCCGT GAACTCAAAA 2760ACTTTCGAGT GGGTGTGGAG TTTCATTAAG TCTAGTAAGT CGAGGGTGAT TATTAGCGGT 2820AAAATAATTC ACAAGGATGT GAATTTGGAC CTCAAGTACG TCGAGAGTTT CGCCGCGGTT 2880ATGTTGGCCT CTGGCGTGCG CAGTAGACTA GCGTCCGAGT ACCTTGCTAA GAACCTTAGT 2940CATTTTTCGG GAGATTGCTC CTTTATTGAA GCCACGTCTT TCGTGTTGCG TGAGAAAATC 3000AGAAACATGA CTCTGAATTT TAACGAAAGA CTTTTACAGT TAGTGAAGCG CGTTGCCTTT 3060GCGACCTTGG ACGTGAGTTT TCTAGATTTA GATTCAACTC TTGAATCAAT AACTGATTTT 3120GCCGAGTGTA AGGTAGCGAT TGAACTCGAC GAGTTGGGTT GCTTGAGAGC GGAGGCCGAG 3180AATGAAAAAA TCAGGAATCT GGCGGGAGAT TCGATTGCGG CTAAACTCGC GAGCGAGATA 3240GTGGTCGATA TTGACTCTAA GCCTTCACCG AAGCAGGTGG GTAATTCGTC ATCCGAAAAC 3300GCCGATAAGC GGGAAGTTCA GAGGCCCGGT TTGCGTGGTG GTTCTAGAAA CGGGGTTGTT 3360GGGGAGTTCC TTCACTTCGT CGTGGATTCT GCCTTGCGTC TTTTCAAATA CGCGACGGAT 3420CAACAACGGA TCAAGTCTTA CGTGCGTTTC TTGGACTCGG CGGTCTCATT CTTGGATTAC 3480AACTACGATA ATCTATCGTT TATACTGCGA GTGCTTTCGG AAGGTTATTC GTGTATGTTC 3540GCGTTTTTGG CGAATCGCGG CGACTTATCT AGTCGTGTCC GTAGCGCGGT GTGTGCTGTG 3600AAAGAAGTTG CTACCTCATG CGCGAACGCG AGCGTTTCTA AAGCCAAGGT TATGATTACC 3660TTCGCAGCGG CCGTGTGTGC TATGATGTTT AATAGCTGCG GTTTTTCAGG CGACGGTCGG 3720GAGTATAAAT CGTATATACA TCGTTACACG CAAGTATTGT TTGACACTAT CTTTTTTGAG 3780GACAGCAGTT ACCTACCCAT AGAAGTTCTG AGTTCGGCGA TATGCGGTGC TATCGTCACA 3840CTTTTCTCCT CGGGCTCGTC CATAAGTTTA AACGCCTTCT TACTTCAAAT TACCAAAGGA 3900TTCTCCCTAG AGGTTGTCGT CCGGAATGTT GTGCGAGTCA CGCATGGTTT GAGCACCACA 3960GCGACCGACG GCGTCATACG TGGGGTTTTC TCCCAAATTG TGTCTCACTT ACTTGTTGGA 4020AATACGGGTA ATGTGGCTTA CCAGTCAGCT TTCATTGCCG GGGTGGTGCC TCTTTTAGTT 4080AAAAAGTGTG TGAGCTTAAT CTTCATCTTG CGTGAAGATA CTTATTCCGG TTTTATTAAG 4140CACGGAATCA GTGAATTCTC TTTCCTTAGT AGTATTCTGA AGTTCTTGAA GGGTAAGCTT 4200GTGGACGAGT TGAAATCGAT TATTCAAGGG GTTTTTGATT CCAACAAGCA CGTGTTTAAA 4260GAAGCTACTC AGGAAGCGAT TCGTACGACG GTCATGCAAG TGCCTGTCGC TGTAGTGGAT 4320GCCCTTAAGA GCGCCGGGGG AAAAATTTAT AACAATTTTA CTAGTCGACG TACCTTTGGT 4380AAGGATGAAG GCTCGTCTAG CGACGGCGCA TGTGAAGAGT ATTTCTCATG CGACGAAGGT 4440GAAGGTCCGG GTCTGAAAGG GGGTTCCAGC TATGGCTTCT CAATTTTAGC GTTCTTTTCA 4500CGCATTATGT GGGGAGCTCG TCGGCTTATT GTTAAGGTGA AGCATGAGTG TTTTGGGAAA 4560CTTTTTGAAT TTCTATCGCT GAAGCTTCAC GAATTCAGGA CTCGCGTTTT TGGGAAGAAT 4620AGAACGGACG TGGGAGTTTA CGATTTTTTG CCCACGGGCA TCGTGGAAAC GCTCTCATCG 4680ATAGAAGAGT GCGACCAAAT TGAAGAACTT CTCGGCGACG ACCTGAAAGG TGACAAGGAT 4740GCTTCGTTGA CCGATATGAA TTACTTTGAG TTCTCAGAAG ACTTCTTAGC CTCTATCGAG 4800GAGCCGCCTT TCGCTGGATT GCGAGGAGGT AGCAAGAACA TCGCGATTTT GGCGATTTTG 4860GAATACGCGG ATAATTTGTT TCGCATTGTC GCAAGCAAGT GTTCGAAACG ACCTTTATTT 4920CTTGCTTTCG CCGAACTCTC AAGCGCCCTT ATCGAGAAAT TTAAGGAGGT TTTCCCTCGT 4980AAGAGCCAGC TCGTCGCTAT CGTGCGCGAG TATACTCAGA GATTCCTCCG AAGTCGCATG 5040CGTGCGTTGG GTTTGAATAA CGAGTTCGTG GTAAAATCTT TCGCCGATTT GCTACCCGCA 5100TTAATGAAGC GGAAGGTTTC AGGTTCGTTC TTAGCTAGTG TTTATCGCCC ACTTAGAGGT 5160TTCTCATATA TGTGTGTTTC AGCGGAGCGA CGTGAAAAGT TTTTTGCTCT CGTGTGTTTA 5220ATCGGGTTAA GTCTCCCTTT CTTCGTGCGC ATCGTAGGAG CGAAAGCGTG CGAAGAACTC 5280GTGTCCTCAG CGCGTCGCTT TTATGAGCGT ATTAAAATTT TTCTAAGGCA GAAGTATGTC 5340TCTCTTTCTA ATTTCTTTTG TCACTTGTTT AGCTCTGACG TTGATGACAG TTCCGCATCT 5400GCAGGGTTGA AAGGTGGTGC GTCGCGAATG ACGCTCTTCC ACCTTCTGGT TCGCCTTGCT 5460AGTGCCCTCC TATCGTTAGG GTGGGAAGGG TTAAAGCTAC TCTTATCGCA CCACAACTTG 5520TTATTTTTGT GTTTTGCATT GGTTGACGAT GTGAACGTCC TTATCAAAGT TCTTGGGGGT 5580CTTTCTTTCT TTGTGCAACC AATCTTTTCC TTGTTTGCGG CGATGCTTCT ACAACCGGAC 5640AGGTTTGTGG AGTATTCCGA GAAACTTGTT ACAGCGTTTG AATTTTTCTT AAAATGTTCG 5700CCTCGCGCGC CTGCACTACT CAAAGGGTTT TTTGAGTGCG TGGCGAACAG CACTGTGTCA 5760AAAACCGTTC GAAGACTTCT TCGCTGTTTC GTGAAGATGC TCAAACTTCG AAAAGGGCGA 5820GGGTTGCGTG CGGATGGTAG GGGTCTCCAT CGGCAGAAAG CCGTACCCGT CATACCTTCT 5880AATCGGGTCG TGACCGACGG GGTTGAAAGA CTTTCGGTAA AGATGCAAGG AGTTGAAGCG 5940TTGCGTACCG AATTGAGAAT CTTAGAAGAT TTAGATTCTG CCGTGATCGA AAAACTCAAT 6000AGACGCAGAA ATCGTGACAC TAATGACGAC GAATTTACGC GCCCTGCTCA TGAGCAGATG 6060CAAGAAGTCA CCACTTTCTG TTCGAAAGCC AACTCTGCTG GTTTGGCCCT GGAAAGGGCA 6120GTGCTTGTGG AAGACGCTAT AAAGTCGGAG AAACTTTCTA AGACGGTTAA TGAGATGGTG 6180AGGAAAGGGA GTACCACCAG CGAAGAAGTG GCCGTCGCTT TGTCGGACGA TGAAGCCGTG 6240GAAGAAATCT CTGTTGCTGA CGAGCGAGAC GATTCGCCTA AGACAGTCAG GATAAGCGAA 6300TACCTAAATA GGTTAAACTC AAGCTTCGAA TTCCCGAAGC CTATTGTTGT GGACGACAAC 6360AAGGATACCG GGGGTCTAAC GAACGCCGTG AGGGAGTTTT ATTATATGCA AGAACTTGCT 6420CTTTTCGAAA TCCACAGCAA ACTGTGCACC TACTACGATC AACTGCGCAT AGTCAACTTC 6480GATCGTTCCG TAGCACCATG CAGCGAAGAT GCTCAGCTGT ACGTACGGAA GAACGGCTCA 6540ACGATAGTGC AGGGTAAAGA GGTACGTTTG CACATTAAGG ATTTCCACGA TCACGATTTC 6600CTGTTTGACG GAAAAATTTC TATTAACAAG CGGCGGCGAG GCGGAAATGT TTTATATCAC 6660GACAACCTCG CGTTCTTGGC GAGTAATTTG TTCTTAGCCG GCTACCCCTT TTCAAGGAGC 6720TTCGTCTTCA CGAATTCGTC GGTCGATATT CTCCTCTACG AAGCTCCACC CGGAGGTGGT 6780AAGACGACGA CGCTGATTGA CTCGTTCTTG AAGGTCTTCA AGAAAGGTGA GGTTTCCACC 6840ATGATCTTAA CCGCCAACAA AAGTTCGCAG GTTGAGATCC TAAAGAAAGT GGAGAAGGAA 6900GTGTCTAACA TTGAATGCCA GAAACGTAAA GACAAAAGAT CTCCGAAAAA GAGCATTTAC 6960ACCATCGACG CTTATTTAAT GCATCACCGT GGTTGTGATG CAGACGTTCT TTTCATCGAT 7020GAGTGTTTCA TGGTTCATGC GGGTAGCGTA CTAGCTTGCA TTGAGTTCAC GAGGTGTCAT 7080AAAGTAATGA TCTTCGGGGA TAGCCGGCAG ATTCACTACA TTGAAAGGAA CGAATTGGAC 7140AAGTGTTTGT ATGGGGATCT CGACAGGTTC GTGGACCTGC AGTGTCGGGT TTATGGTAAT 7200ATTTCGTACC GTTGTCCATG GGATGTGTGC GCTTGGTTAA GCACAGTGTA TGGCAACCTA 7260ATCGCCACCG TGAAGGGTGA AAGCGAAGGT AAGAGCAGCA TGCGCATTAA CGAAATTAAT 7320TCAGTCGACG ATTTAGTCCC CGACGTGGGT TCCACGTTTC TGTGTATGCT TCAGTCGGAG 7380AAGTTGGAAA TCAGCAAGCA CTTTATTCGC AAGGGTTTGA CTAAACTTAA CGTTCTAACG 7440GTGCATGAGG CGCAAGGTGA GACGTATGCG CGTGTGAACC TTGTGCGACT TAAGTTTCAG 7500GAGGATGAAC CCTTTAAATC TATCAGGCAC ATAACCGTCG CTCTTTCTCG TCACACCGAC 7560AGCTTAACTT ATAACGTCTT AGCTGCTCGT CGAGGTGACG CCACTTGCGA TGCCATCCAG 7620AAGGCTGCGG AATTGGTGAA CAAGTTTCGC GTTTTTCCTA CATCTTTTGG TGGTAGTGTT 7680ATCAATCTCA ACGTGAAGAA GGACGTGGAA GATAACAGTA GGTGCAAGGC TTCGTCGGCA 7740CCATTGAGCG TAATCAACGA CTTTTTGAAC GAAGTTAATC CCGGTACTGC GGTGATTGAT 7800TTTGGTGATT TGTCCGCGGA CTTCAGTACT GGGCCTTTTG AGTGCGGTGC CAGCGGTATT 7860GTGGTGCGGG ACAACATCTC CTCCAGCAAC ATCACTGATC ACGATAAGCA GCGTGTTTAG 7920

The large polyprotein (papain-like proteases, methyltransferase, andhelicase) has an amino acid sequence corresponding to SEQ. ID. No. 3 asfollows: Thr Leu Arg Glu Asn Pro Ile Ser Val Ser Gly Val Asn Leu Gly Arg1               5                   10                  15 Ser Ala AlaAla Gln Val Ile Tyr Phe Gly Ser Phe Thr Gln Pro Phe            20                  25                  30 Ala Leu Tyr ProArg Gln Glu Ser Ala Ile Val Lys Thr Gln Leu Pro        35                  40                  45 Pro Val Ser Val ValLys Val Glu Cys Val Ala Ala Glu Val Ala Pro    50                  55                  60 Asp Arg Gly Val Val AspLys Lys Pro Thr Ser Val Gly Val Pro Pro65                  70                  75                  80 Gln ArgGly Val Leu Ser Phe Pro Thr Val Val Arg Asn Arg Gly Asp                85                  90                  95 Val Ile IleThr Gly Val Val His Glu Ala Leu Lys Lys Ile Lys Asp            100                 105                 110 Gly Leu Leu ArgPhe Arg Val Gly Gly Asp Met Arg Phe Ser Arg Phe        115                 120                 125 Phe Ser Ser Asn TyrGly Cys Arg Phe Val Ala Ser Val Arg Thr Asn    130                 135                 140 Thr Thr Val Trp Leu AsnCys Thr Lys Ala Ser Gly Glu Lys Phe Ser145                 150                 155                 160 Leu AlaAla Ala Cys Thr Ala Asp Tyr Val Ala Met Leu Arg Tyr Val                165                 170                 175 Cys Gly GlyLys Phe Pro Leu Val Leu Met Ser Arg Val Ile Tyr Pro            180                 185                 190 Asp Gly Arg CysTyr Leu Ala His Met Arg Tyr Leu Cys Ala Phe Tyr        195                 200                 205 Cys Arg Pro Phe ArgGlu Ser Asp Tyr Ala Leu Gly Met Trp Pro Thr    210                 215                 220 Val Ala Arg Leu Arg AlaCys Val Glu Lys Asn Phe Gly Val Glu Ala225                 230                 235                 240 Cys GlyIle Ala Leu Arg Gly Tyr Tyr Thr Ser Arg Asn Val Tyr His                245                 250                 255 Cys Asp TyrAsp Ser Ala Tyr Val Lys Tyr Phe Arg Asn Leu Ser Gly            260                 265                 270 Arg Ile Gly GlyGly Ser Phe Asp Pro Thr Ser Leu Thr Ser Val Ile        275                 280                 285 Thr Val Lys Ile SerGly Leu Pro Gly Gly Leu Pro Lys Asn Ile Ala    290                 295                 300 Phe Gly Ala Phe Leu CysAsp Ile Arg Tyr Val Glu Pro Val Asp Ser305                 310                 315                 320 Gly GlyIle Gln Ser Ser Val Lys Thr Lys Arg Glu Asp Ala His Arg                325                 330                 335 Thr Val GluGlu Arg Ala Ala Gly Gly Ser Val Glu Gln Pro Arg Gln            340                 345                 350 Lys Arg Ile AspGlu Lys Gly Cys Gly Arg Val Pro Ser Gly Gly Phe        355                 360                 365 Ser His Leu Leu ValGly Asn Leu Asn Glu Val Arg Arg Lys Val Ala    370                 375                 380 Ala Gly Leu Leu Arg PheArg Val Gly Gly Asp Met Asp Phe His Arg385                 390                 395                 400 Ser PheSer Thr Gln Ala Gly His Arg Leu Leu Val Trp Arg Arg Ser                405                 410                 415 Ser Arg SerVal Cys Leu Glu Leu Tyr Ser Pro Ser Lys Asn Phe Leu            420                 425                 430 Arg Tyr Asp ValLeu Pro Cys Ser Gly Asp Tyr Ala Ala Met Phe Ser        435                 440                 445 Phe Ala Ala Gly GlyArg Phe Pro Leu Val Leu Met Thr Arg Ile Arg    450                 455                 460 Tyr Pro Asn Gly Phe CysTyr Leu Ala His Cys Arg Tyr Ala Cys Ala465                 470                 475                 480 Phe LeuLeu Arg Gly Phe Asp Pro Lys Arg Phe Asp Ile Gly Ala Phe                485                 490                 495 Pro Thr AlaAla Lys Leu Arg Asn Arg Met Val Ser Glu Leu Gly Glu            500                 505                 510 Arg Ser Leu GlyLeu Asn Leu Tyr Gly Ala Tyr Thr Ser Arg Gly Val        515                 520                 525 Phe His Cys Asp TyrAsp Ala Lys Phe Ile Lys Asp Leu Arg Leu Met    530                 535                 540 Ser Ala Val Ile Ala GlyLys Asp Gly Val Glu Glu Val Val Pro Ser545                 550                 555                 560 Asp IleThr Pro Ala Met Lys Gln Lys Thr Ile Glu Ala Val Tyr Asp                565                 570                 575 Arg Leu TyrGly Gly Thr Asp Ser Leu Leu Lys Leu Ser Ile Glu Lys            580                 585                 590 Asp Leu Ile AspPhe Lys Asn Asp Val Gln Ser Leu Lys Lys Asp Arg        595                 600                 605 Pro Ile Val Lys ValPro Phe Tyr Met Ser Glu Ala Thr Gln Asn Ser    610                 615                 620 Leu Thr Arg Phe Tyr ProGln Phe Glu Leu Lys Phe Ser His Ser Ser625                 630                 635                 640 His SerAsp His Pro Ala Ala Ala Ala Ser Arg Leu Leu Glu Asn Glu                645                 650                 655 Thr Leu ValArg Leu Cys Gly Asn Ser Val Ser Asp Ile Gly Gly Cys            660                 665                 670 Pro Leu Phe HisLeu His Ser Lys Thr Gln Arg Arg Val His Val Cys        675                 680                 685 Arg Pro Val Leu AspGly Lys Asp Ala Gln Arg Arg Val Val Arg Asp    690                 695                 700 Leu Gln Tyr Ser Asn ValArg Leu Gly Asp Asp Asp Lys Ile Leu Glu705                 710                 715                 720 Gly ProArg Asn Ile Asp Ile Cys His Tyr Pro Leu Gly Ala Cys Asp                725                 730                 735 His Glu SerSer Ala Met Met Met Val Gln Val Tyr Asp Ala Ser Leu            740                 745                 750 Tyr Glu Ile CysGly Ala Met Ile Lys Lys Lys Ser Arg Ile Thr Tyr        755                 760                 765 Leu Thr Met Val ThrPro Gly Glu Phe Leu Asp Gly Arg Glu Cys Val    770                 775                 780 Tyr Met Glu Ser Leu AspCys Glu Ile Glu Val Asp Val His Ala Asp785                 790                 795                 800 Val ValMet Tyr Lys Phe Gly Ser Ser Cys Tyr Ser His Lys Leu Ser                805                 810                 815 Ile Ile LysAsp Ile Met Thr Thr Pro Tyr Leu Thr Leu Gly Gly Phe            820                 825                 830 Leu Phe Ser ValGlu Met Tyr Glu Val Arg Met Gly Val Asn Tyr Phe        835                 840                 845 Lys Ile Thr Lys SerGlu Val Ser Pro Ser Ile Ser Cys Thr Lys Leu    850                 855                 860 Leu Arg Tyr Arg Arg AlaAsn Ser Asp Val Val Lys Val Lys Leu Pro865                 870                 875                 880 Arg PheAsp Lys Lys Arg Arg Met Cys Leu Pro Gly Tyr Asp Thr Ile                885                 890                 895 Tyr Leu AspSer Lys Phe Val Ser Arg Val Phe Asp Tyr Val Val Cys            900                 905                 910 Asn Cys Ser AlaVal Asn Ser Lys Thr Phe Glu Trp Val Trp Ser Phe        915                 920                 925 Ile Lys Ser Ser LysSer Arg Val Ile Ile Ser Gly Lys Ile Ile His    930                 935                 940 Lys Asp Val Asn Leu AspLeu Lys Tyr Val Glu Ser Phe Ala Ala Val945                 950                 955                 960 Met LeuAla Ser Gly Val Arg Ser Arg Leu Ala Ser Glu Tyr Leu Ala                965                 970                 975 Lys Asn LeuSer His Phe Ser Gly Asp Cys Ser Phe Ile Glu Ala Thr            980                 985                 990 Ser Phe Val LeuArg Glu Lys Ile Arg Asn Met Thr Leu Asn Phe Asn        995                 1000                1005 Glu Arg Leu Leu GlnLeu Val Lys Arg Val Ala Phe Ala Thr Leu Asp    1010                1015                1020 Val Ser Phe Leu Asp LeuAsp Ser Thr Leu Glu Ser Ile Thr Asp Phe1025                1030                1035                1040 Ala GluCys Lys Val Ala Ile Glu Leu Asp Glu Leu Gly Cys Leu Arg                1045                1050                1055 Ala Glu AlaGlu Asn Glu Lys Ile Arg Asn Leu Ala Gly Asp Ser Ile            1060                1065                1070 Ala Ala Lys LeuAla Ser Glu Ile Val Val Asp Ile Asp Ser Lys Pro        1075                1080                1085 Ser Pro Lys Gln ValGly Asn Ser Ser Ser Glu Asn Ala Asp Lys Arg    1090                1095                1100 Glu Val Gln Arg Pro GlyLeu Arg Gly Gly Ser Arg Asn Gly Val Val1105                1110                1115                1120 Gly GluPhe Leu His Phe Val Val Asp Ser Ala Leu Arg Leu Phe Lys                1125                1130                1135 Tyr Ala ThrAsp Gln Gln Arg Ile Lys Ser Tyr Val Arg Phe Leu Asp            1140                1145                1150 Ser Ala Val SerPhe Leu Asp Tyr Asn Tyr Asp Asn Leu Ser Phe Ile        1155                1160                1165 Leu Arg Val Leu SerGlu Gly Tyr Ser Cys Met Phe Ala Phe Leu Ala    1170                1175                1180 Asn Arg Gly Asp Leu SerSer Arg Val Arg Ser Ala Val Cys Ala Val1185                1190                1195                1200 Lys GluVal Ala Thr Ser Cys Ala Asn Ala Ser Val Ser Lys Ala Lys                1205                1210                1215 Val Met IleThr Phe Ala Ala Ala Val Cys Ala Met Met Phe Asn Ser            1220                1225                1230 Cys Gly Phe SerGly Asp Gly Arg Glu Tyr Lys Ser Tyr Ile His Arg        1235                1240                1245 Tyr Thr Gln Val LeuPhe Asp Thr Ile Phe Phe Glu Asp Ser Ser Tyr    1250                1255                1260 Leu Pro Ile Glu Val LeuSer Ser Ala Ile Cys Gly Ala Ile Val Thr1265                1270                1275                1280 Leu PheSer Ser Gly Ser Ser Ile Ser Leu Asn Ala Phe Leu Leu Gln                1285                1290                1295 Ile Thr LysGly Phe Ser Leu Glu Val Val Val Arg Asn Val Val Arg            1300                1305                1310 Val Thr His GlyLeu Ser Thr Thr Ala Thr Asp Gly Val Ile Arg Gly        1315                1320                1325 Val Phe Ser Gln IleVal Ser His Leu Leu Val Gly Asn Thr Gly Asn    1330                1335                1340 Val Ala Tyr Gln Ser AlaPhe Ile Ala Gly Val Val Pro Leu Leu Val1345                1350                1355                1360 Lys LysCys Val Ser Leu Ile Phe Ile Leu Arg Glu Asp Thr Tyr Ser                1365                1370                1375 Gly Phe IleLys His Gly Ile Ser Glu Phe Ser Phe Leu Ser Ser Ile            1380                1385                1390 Leu Lys Phe LeuLys Gly Lys Leu Val Asp Glu Leu Lys Ser Ile Ile        1395                1400                1405 Gln Gly Val Phe AspSer Asn Lys His Val Phe Lys Glu Ala Thr Gln    1410                1415                1420 Glu Ala Ile Arg Thr ThrVal Met Gln Val Pro Val Ala Val Val Asp1425                1430                1435                1440 Ala LeuLys Ser Ala Ala Gly Lys Ile Tyr Asn Asn Phe Thr Ser Arg                1445                1450                1455 Arg Thr PheGly Lys Asp Glu Gly Ser Ser Ser Asp Gly Ala Cys Glu            1460                1465                1470 Glu Tyr Phe SerCys Asp Glu Gly Glu Gly Pro Gly Leu Lys Gly Gly        1475                1480                1485 Ser Ser Tyr Gly PheSer Ile Leu Ala Phe Phe Ser Arg Ile Met Trp    1490                1495                1500 Gly Ala Arg Arg Leu IleVal Lys Val Lys His Glu Cys Phe Gly Lys1505                1510                1515                1520 Leu PheGlu Phe Leu Ser Leu Lys Leu His Glu Phe Arg Thr Arg Val                1525                1530                1535 Phe Gly LysAsn Arg Thr Asp Val Gly Val Tyr Asp Phe Leu Pro Thr            1540                1545                1550 Gly Ile Val GluThr Leu Ser Ser Ile Glu Glu Cys Asp Gln Ile Glu        1555                1560                1565 Glu Leu Leu Gly AspAsp Leu Lys Gly Asp Lys Asp Ala Ser Leu Thr    1570                1575                1580 Asp Met Asn Tyr Phe GluPhe Ser Glu Asp Phe Leu Ala Ser Ile Glu1585                1590                1595                1600 Glu ProPro Phe Ala Gly Leu Arg Gly Gly Ser Lys Asn Ile Ala Ile                1605                1610                1615 Leu Ala IleLeu Glu Tyr Ala His Asn Leu Phe Arg Ile Val Ala Ser            1620                1625                1630 Lys Cys Ser LysArg Pro Leu Phe Leu Ala Phe Ala Glu Leu Ser Ser        1635                1640                1645 Ala Leu Ile Glu LysPhe Lys Glu Val Phe Pro Arg Lys Ser Gln Leu    1650                1655                1660 Val Ala Ile Val Arg GluTyr Thr Gln Arg Phe Leu Arg Ser Arg Met1665                1670                1675                1680 Arg AlaLeu Gly Leu Asn Asn Glu Phe Val Val Lys Ser Phe Ala Asp                1685                1690                1695 Leu Leu ProAla Leu Met Lys Arg Lys Val Ser Gly Ser Phe Leu Ala            1700                1705                1710 Ser Val Tyr ArgPro Leu Arg Gly Phe Ser Tyr Met Cys Val Ser Ala        1715                1720                1725 Glu Arg Arg Glu LysPhe Phe Ala Leu Val Cys Leu Ile Gly Leu Ser    1730                1735                1740 Leu Pro Phe Phe Val ArgIle Val Gly Ala Lys Ala Cys Glu Glu Leu1745                1750                1755                1760 Val SerSer Ala Arg Arg Phe Tyr Glu Arg Ile Lys Ile Phe Leu Arg                1765                1770                1775 Gln Lys TyrVal Ser Leu Ser Asn Phe Phe Cys His Leu Phe Ser Ser            1780                1785                1790 Asp Val Asp AspSer Ser Ala Ser Ala Gly Leu Lys Gly Gly Ala Ser                1795                1800                1805 Arg Met ThrLeu Phe His Leu Leu Val Arg Leu Ala Ser Ala Leu Leu    1810                1815                1820 Ser Leu Gly Trp Glu GlyLeu Lys Leu Leu Leu Ser His His Asn Leu1825                1830                1835                1840 Leu PheLeu Cys Phe Ala Leu Val Asp Asp Val Asn Val Leu Ile Lys                1845                1850                1855 Val Leu GlyGly Leu Ser Phe Phe Val Gln Pro Ile Phe Ser Leu Phe            1860                1865                1870 Ala Ala Met LeuLeu Gln Pro Asp Arg Phe Val Glu Tyr Ser Glu Lys        1875                1880                1885 Leu Val Thr Ala PheGlu Phe Phe Leu Lys Cys Ser Pro Arg Ala Pro    1890                1895                1900 Ala Leu Leu Lys Gly PhePhe Glu Cys Val Ala Asn Ser Thr Val Ser1905                1910                1915                1920 Lys ThrVal Arg Arg Leu Leu Arg Cys Phe Val Lys Met Leu Lys Leu                1925                1930                1935 Arg Lys GlyArg Gly Leu Arg Ala Asp Gly Arg Gly Leu His Arg Gln            1940                1945                1950 Lys Ala Val ProVal Ile Pro Ser Asn Arg Val Val Thr Asp Gly Val        1955                1960                1965 Glu Arg Leu Ser ValLys Met Gln Gly Val Glu Ala Leu Arg Thr Glu    1970                1975                1980 Leu Arg Ile Leu Glu AspLeu Asp Ser Ala Val Ile Glu Lys Leu Asn1985                1990                1995                2000 Arg ArgArg Asn Arg Asp Thr Asn Asp Asp Glu Phe Thr Arg Pro Ala                2005                2010                2015 His Glu GlnMet Gln Glu Val Thr Thr Phe Cys Ser Lys Ala Asn Ser            2020                2025                2030 Ala Gly Leu AlaLeu Glu Arg Ala Val Leu Val Glu Asp Ala Ile Lys        2035                2040                2045 Ser Glu Lys Leu SerLys Thr Val Asn Glu Met Val Arg Lys Gly Ser    2050                2055                2060 Thr Thr Ser Glu Glu ValAla Val Ala Leu Ser Asp Asp Glu Ala Val2065                2070                2075                2080 Glu GluIle Ser Val Ala Asp Glu Arg Asp Asp Ser Pro Lys Thr Val                2085                2090                2095 Arg Ile SerGlu Tyr Leu Asn Arg Leu Asn Ser Ser Phe Glu Phe Pro            2100                2105                2110 Lys Pro Ile ValVal Asp Asp Asn Lys Asp Thr Gly Gly Leu Thr Asn        2115                2120                2125 Ala Val Arg Glu PheTyr Tyr Met Gln Glu Leu Ala Leu Phe Glu Ile    2130                2135                2140 His Ser Lys Leu Cys ThrTyr Tyr Asp Gln Leu Arg Ile Val Asn Phe2145                2150                2155                2160 Asp ArgSer Val Ala Pro Cys Ser Glu Asp Ala Gln Leu Tyr Val Arg                2165                2170                2175 Lys Asn GlySer Thr Ile Val Gln Gly Lys Glu Val Arg Leu His Ile            2180                2185                2190 Lys Asp Phe HisAsp His Asp Phe Leu Phe Asp Gly Lys Ile Ser Ile        2195                2200                2205 Asn Lys Arg Arg ArgGly Gly Asn Val Leu Tyr His Asp Asn Leu Ala    2210                2215                2220 Phe Leu Ala Ser Asn LeuPhe Leu Ala Gly Tyr Pro Phe Ser Arg Ser2225                2230                2235                2240 Phe ValPhe Thr Asn Ser Ser Val Asp Ile Leu Leu Tyr Glu Ala Pro                2245                2250                2255 Pro Gly GlyGly Lys Thr Thr Thr Leu Ile Asp Ser Phe Leu Lys Val            2260                2265                2270 Phe Lys Lys GlyGlu Val Ser Thr Met Ile Leu Thr Ala Asn Lys Ser        2275                2280                2285 Ser Gln Val Glu IleLeu Lys Lys Val Glu Lys Glu Val Ser Asn Ile    2290                2295                2300 Glu Cys Gln Lys Arg LysAsp Lys Arg Ser Pro Lys Lys Ser Ile Tyr2305                2310                2315                2320 Thr IleAsp Ala Tyr Leu Met His His Arg Gly Cys Asp Ala Asp Val                2325                2330                2335 Leu Phe IleAsp Glu Cys Phe Met Val His Ala Gly Ser Val Leu Ala            2340                2345                2350 Cys Ile Glu PheThr Arg Cys His Lys Val Met Ile Phe Gly Asp Ser        2355                2360                2365 Arg Gln Ile His TyrIle Glu Arg Asn Glu Leu Asp Lys Cys Leu Tyr    2370                2375                2380 Gly Asp Leu Asp Arg PheVal Asp Leu Gln Cys Arg Val Tyr Gly Asn2385                2390                2395                2400 Ile SerTyr Arg Cys Pro Trp Asp Val Cys Ala Trp Leu Ser Thr Val                2405                2410                2415 Tyr Gly AsnLeu Ile Ala Thr Val Lys Gly Glu Ser Glu Gly Lys Ser            2420                2425                2430 Ser Met Arg IleAsn Glu Ile Asn Ser Val Asp Asp Leu Val Pro Asp        2435                2440                2445 Val Gly Ser Thr PheLeu Cys Met Leu Gln Ser Glu Lys Leu Glu Ile    2450                2455                2460 Ser Lys His Phe Ile ArgLys Gly Leu Thr Lys Leu Asn Val Leu Thr2465                2470                2475                2480 Val HisGlu Ala Gln Gly Glu Thr Tyr Ala Arg Val Asn Leu Val Arg                2485                2490                2495 Leu Lys PheGln Glu Asp Glu Pro Phe Lys Ser Ile Arg His Ile Thr            2500                2505                2510 Val Ala Leu SerArg His Thr Asp Ser Leu Thr Tyr Asn Val Leu Ala        2515                2520                2525 Ala Arg Arg Gly AspAla Thr Cys Asp Ala Ile Gln Lys Ala Ala Glu    2530                2535                2540 Leu Val Asn Lys Phe ArgVal Phe Pro Thr Ser Phe Gly Gly Ser Val2545                2550                2555                2560 Ile AsnLeu Asn Val Lys Lys Asp Val Glu Asp Asn Ser Arg Cys Lys                2565                2570                2575 Ala Ser SerAla Pro Leu Ser Val Ile Asn Asp Phe Leu Asn Glu Val            2580                2585                2590 Asn Pro Gly ThrAla Val Ile Asp Phe Gly Asp Leu Ser Ala Asp Phe        2595                2600                2605 Ser Thr Gly Pro PheGlu Cys Gly Ala Ser Gly Ile Val Val Arg Asp    2610                2615                2620 Asn Ile Ser Ser Ser AsnIle Thr Asp His Asp Lys Gln Arg Val2625                2630                2635and has a molecular weight of about 290 to 300 kDa, preferably 294 kDa.

Another such DNA molecule (GLRaV-2 ORFlb) includes nucleotides 7922-9301of SEQ. ID. No. 1 and codes for a grapevine leafroll virus RNA-dependentRNA polymerase (RdRP). This DNA molecule comprises the nucleotidesequence corresponding to SEQ. ID. No. 4 as follows: AGCGTAGTTCGGTCGCAGGC GATTCCGCGT AGAAAACCTT CTCTACAAGA AAATTTGTAT   60 TGGTTTGAAGCGCGGAATTA TAACTTCTCG ACTTGCGACC GTAACACATC TGCTTCAATG  120 TTCGGAGAGGCTATGGCGAT GAACTGTCTT CGTCGTTGCT TCGACCTAGA TGCCTTTTCG  180 TCCCTGCGTGATGATGTGAT TAGTATCACA CGTTCAGGCA TCGAACAATG GCTGGAGAAA  240 CGTACTCCTAGTCAGATTAA AGCATTAATG AAGGATGTTG AATCGCCTTT GGAAATTGAC  300 GATGAAATTTGTCGTTTTAA GTTGATGGTG AAGCGTGACG CTAAGGTGAA GTTAGACTCT  360 TCTTGTTTAACTAAACACAG CGCCGCTCAA AATATCATGT TTCATCGCAA GAGCATTAAT  420 GCTATCTTCTCTCCTATCTT TAATGAGGTG AAAAACCGAA TAATGTGCTG TCTTAAGCCT  480 AACATAAAGTTTTTTACGGA GATGACTAAC AGGGATTTTG CTTCTGTTGT CAGCAACATG  540 CTTGGTGACGACGATGTGTA CCATATAGGT GAAGTTGATT TCTCAAAGTA CGACAAGTCT  600 CAAGATGCTTTCGTGAAGGC TTTTGAAGAA GTAATGTATA AGGAACTCGG TGTTGATGAA  660 GAGTTGCTGGCTATCTGGAT GTGCGGCGAG CGGTTATCGA TAGCTAACAC TCTCGATGGT  720 CAGTTGTCCTTCACGATCGA GAATCAAAGG AAGTCGGGAG CTTCGAACAC TTGGATTGGT  780 AACTCTCTCGTCACTTTGGG TATTTTAAGT CTTTACTACG ACGTTAGAAA TTTCGAGGCG  840 TTGTACATCTCGGGCGATGA TTCTTTAATT TTTTCTCGCA GCGAGATTTC GAATTATGCC  900 GAGGACATATGCACTGACAT GGGTTTTGAG ACAAAATTTA TGTCCCCAAG TGTCCCGTAC  960 TTTTGTTCTAAATTTGTTGT TATGTGTGGT CATAAGACGT TTTTTGTTCC CGACCCGTAC 1020 AAGCTTTTTGTCAAGTTGGG AGCAGTCAAA GAGGATGTTT CAATGGATTT CCTTTTCGAG 1080 ACTTTTACCTCCTTTAAAGA CTTAACCTCC GATTTTAACG ACGAGCGCTT AATTCAAAAG 1140 CTCGCTGAACTTGTGGCTTT AAAATATGAG GTTCAAACCG GCAACACCAC CTTGGCGTTA 1200 AGTGTGATACATTGTTTGCG TTCGAATTTC CTCTCGTTTA GCAAGTTATA TCCTCGCGTG 1260 AAGGGATGGCAGGTTTTTTA CACGTCGGTT AAGAAAGCGC TTCTCAAGAG TGGGTGTTCT 1320 CTCTTCGACAGTTTCATGAC CCCTTTTGGT CAGGCTGTCA TGGTTTGGGA TGATGAGTAG 1380

The RNA-dependent RNA polymerase has an amino acid sequencecorresponding to SEQ. ID. No. 5 as follows: Ser Val Val Arg Ser Gln AlaIle Pro Arg Arg Lys Pro Ser Leu Gln1               5                   10                  15 Glu Asn LeuTyr Ser Phe Glu Ala Arg Asn Tyr Asn Phe Ser Thr Cys            20                  25                  30 Asp Arg Asn ThrSer Ala Ser Met Phe Gly Glu Ala Met Ala Met Asn        35                  40                  45 Cys Leu Arg Arg CysPhe Asp Leu Asp Ala Phe Ser Ser Leu Arg Asp    50                  55                  60 Asp Val Ile Ser Ile ThrArg Ser Gly Ile Glu Gln Trp Leu Glu Lys65                  70                  75                  80 Arg ThrPro Ser Gln Ile Lys Ala Leu Met Lys Asp Val Glu Ser Pro                85                  90                  95 Leu Glu IleAsp Asp Glu Ile Cys Arg Phe Lys Leu Met Val Lys Arg            100                 105                 110 Asp Ala Lys ValLys Leu Asp Ser Ser Cys Leu Thr Lys His Ser Ala        115                 120                 125 Ala Gln Asn Ile MetPhe His Arg Lys Ser Ile Asn Ala Ile Phe Ser    130                 135                 140 Pro Ile Phe Asn Glu ValLys Asn Arg Ile Met Cys Cys Leu Lys Pro145                 150                 155                 160 Asn IleLys Phe Phe Thr Glu Met Thr Asn Arg Asp Phe Ala Ser Val                165                 170                 175 Val Ser AsnMet Leu Gly Asp Asp Asp Val Tyr His Ile Gly Glu Val            180                 185                 190 Asp Phe Ser LysTyr Asp Lys Ser Gln Asp Ala Phe Val Lys Ala Phe        195                 200                 205 Glu Glu Val Met TyrLys Glu Leu Gly Val Asp Glu Glu Leu Leu Ala    210                 215                 220 Ile Trp Met Cys Gly GluArg Leu Ser Ile Ala Asn Thr Leu Asp Gly225                 230                 235                 240 Gln LeuSer Phe Thr Ile Glu Asn Gln Arg Lys Ser Gly Ala Ser Asn                245                 250                 255 Thr Trp IleGly Asn Ser Leu Val Thr Leu Gly Ile Leu Ser Leu Tyr            260                 265                 270 Tyr Asp Val ArgAsn Phe Glu Ala Leu Tyr Ile Ser Gly Asp Asp Ser        275                 280                 285 Leu Ile Phe Ser ArgSer Glu Ile Ser Asn Tyr Ala Asp Asp Ile Cys    290                 295                 300 Thr Asp Met Gly Phe GluThr Lys Phe Met Ser Pro Ser Val Pro Tyr305                 310                 315                 320 Phe CysSer Lys Phe Val Val Met Cys Gly His Lys Thr Phe Phe Val                325                 330                 335 Pro Asp ProTyr Lys Leu Phe Val Lys Leu Gly Ala Val Lys Glu Asp            340                 345                 350 Val Ser Met AspPhe Leu Phe Glu Thr Phe Thr Ser Phe Lys Asp Leu        355                 360                 365 Thr Ser Asp Phe AsnAsp Glu Arg Leu Ile Gln Lys Leu Ala Glu Leu    370                 375                 380 Val Ala Leu Lys Tyr GluVal Gln Thr Gly Asn Thr Thr Leu Ala Leu385                 390                 395                 400 Ser ValIle His Cys Leu Arg Ser Asn Phe Leu Ser Phe Ser Lys Leu                405                 410                 415 Tyr Pro ArgVal Lys Gly Trp Gln Val Phe Tyr Thr Ser Val Lys Lys            420                 425                 430 Ala Leu Leu LysSer Gly Cys Ser Leu Phe Asp Ser Phe Met Thr Pro        435                 440                 445 Phe Gly Gln Ala ValMet Val Trp Asp Asp Glu     450                 455and a molecular weight from about 50 to about 54 kDa, preferably about52 kDa.

Another such DNA molecule (GLRAV-2 ORF2) includes nucleotides 9365-9535of SEQ. ID. No. 1 and codes for a small, grapevine leafroll virushydrophobic protein or polypeptide. This DNA molecule comprises thenucleotide sequence corresponding to SEQ. ID. No. 6 as follows:ATGAATCAGG TTTTGCAGTT TGAATGTTTG TTTCTGCTGA ATCTCGCGGT TTTTGCTGTG  60ACTTTCATTT TCATTCTTCT GGTCTTCCGC GTGATTAAGT CTTTTCGCCA GAAGGGTCAC 120GAAGGACCTG TTCCCGTTGT TCGTGGCGGG GGTTTTTCAA CCGTAGTGTA G 171

The small hydrophobic protein or polypeptide has an amino acid sequencecorresponding to SEQ. ID. No. 7 as follows: Met Asn Gln Val Leu Gln PheGlu Cys Leu Phe Leu Leu Asn Leu Ala1               5                   10                  15 Val Phe AlaVal Thr Phe Ile Phe Ile Leu Leu Val Phe Arg Val Ile            20                  25                  30 Lys Ser Phe ArgGln Lys Gly His Glu Ala Pro Val Pro Val Val Arg        35                  40                  45 Gly Gly Gly Phe SerThr Val Val     50                  55and a molecular weight from about 5 to about 7 kDa, preferably about 6kDa.

Another such DNA molecule (GLRaV-2 ORF3) includes nucleotides 9551-11350of SEQ. ID. No. 1 and encodes for a grapevine leafroll virus heat shock70 protein. This DNA molecule comprises the nucleotide sequencecorresponding to SEQ. ID. No. 8 as follows: ATGGTAGTTT TCGGTTTGGACTTTGGCACC ACATTCTCTA CGGTGTGTGT GTACAAGGAT   60 GGACGAGTTT TTTCATTCAAGCAGAATAAT TCGGCGTACA TCCCCACTTA CCTCTATCTC  120 TTCTCCGATT CTAACCACATGACTTTTGGT TACGAGGCCG AATCACTGAT GAGTAATCTG  180 AAAGTTAAAG GTTCGTTTTATAGAGATTTA AAACGTTGGG TGGGTTGCGA TTCGAGTAAC  240 CTCGACGCGT ACCTTGACCGTTTAAAACCT CATTACTCGG TCCGCTTGGT TAAGATCGGC  300 TCTGGCTTGA ACGAAACTGTTTCAATTGGA AACTTCGGGG GCACTGTTAA GTCTGAGGCT  360 CATCTGCCAG GGTTGATAGCTCTCTTTATT AAGGCTGTCA TTAGTTGCGC GGAGGGCGCG  420 TTTGCGTGCA CTTGCACCGGGGTTATTTGT TCAGTACCTG CCAATTATGA TAGCGTTCAA  480 AGGAATTTCA CTGATCAGTGTGTTTCACTC AGCGGTTATC AGTGCGTATA TATGATCAAT  540 GAACCTTCAG CGGCTGCGCTATCTGCGTGT AATTCGATTG GAAAGAAGTC CGCAAATTTG  600 GCTGTTTACG ATTTCGGTGGTGGGACCTTC GACGTGTCTA TCATTTCATA CCGCAACAAT  660 ACTTTTGTTG TGCGAGCTTCTGGAGGCGAT CTAAATCTCG GTGGAAGGGA TGTTGATCGT  720 GCGTTTCTCA CGCACCTCTTCTCTTTAACA TCGCTGGAAC CTGACCTCAC TTTGGATATC  780 TCGAATCTGA AAGAATCTTTATCAAAAACG GACGCAGAGA TAGTTTACAC TTTGAGAGGT  840 GTCGATGGAA GAAAAGAAGACGTTAGAGTA AACAAAAACA TTCTTACGTC GGTGATGCTC  900 CCCTACGTGA ACAGAACGCTTAAGATATTA GAGTCAACCT TAAAATCGTA TGCTAAGAGT  960 ATGAATGAGA GTGCGCGAGTTAAGTGCGAT TTAGTGCTGA TAGGAGGATC TTCATATCTT 1020 CCTGGCCTGG CAGACGTACTAACGAAGCAT CAGAGCGTTG ATCGTATCTT AAGAGTTTCG 1080 GATCCTCGGG CTGCCGTGGCCGTCGGTTGC GCATTATATT CTTCATGCCT CTCAGGATCT 1140 GGGGGGTTGC TACTGATCGACTGTGCAGCT CACACTGTCG CTATAGCGGA CAGAAGTTGT 1200 CATCAAATCA TTTGCGCTCCAGCGGGGGCA CCGATCCCCT TTTCAGGAAG CATGCCTTTG 1260 TACTTAGCCA GGGTCAACAAGAACTCGCAG CGTGAAGTCG CCGTGTTTGA AGGGGAGTAC 1320 GTTAAGTGCC CTAAGAACAGAAAGATCTGT GGAGCAAATA TAAGATTTTT TGATATAGGA 1380 GTGACGGGTG ATTCGTACGCACCCGTTACC TTCTATATGG ATTTCTCCAT TTCAAGCGTA 1440 GGAGCCGTTT CATTCGTGGTGAGAGGTCCT GAGGGTAAGC AAGTGTCACT CACTGGAACT 1500 CCAGCGTATA ACTTTTCGTCTGTGGCTCTC GGATCACGCA GTGTCCGAGA ATTGCATATT 1560 AGTTTAAATA ATAAAGTTTTTCTCGGTTTG CTTCTACATA GAAAGGCGGA TCGACGAATA 1620 CTTTTCACTA AGGATGAAGCGATTCGATAC GCCGATTCAA TTGATATCGC GGATGTGCTA 1680 AAGGAATATA AAAGTTACGCGGCCAGTGCC TTACCACCAG ACGAGGATGT CGAATTACTC 1740 CTGGGAAAGT CTGTTCAAAAAGTTTTACGG GGAAGCAGAC TGGAAGAAAT ACCTCTCTAG 1800

The heat shock 70 protein is believed to function as a chaperone proteinand has an amino acid sequence corresponding to SEQ. ID. No. 9 asfollows: Met Val Val Phe Gly Leu Asp Phe Gly Thr Thr Phe Ser Thr Val Cys1               5                   10                  15 Val Tyr LysAsp Gly Arg Val Phe Ser Phe Lys Gln Asn Asn Ser Ala            20                  25                  30 Tyr Ile Pro ThrTyr Leu Tyr Leu Phe Ser Asp Ser Asn His Met Thr        35                  40                  45 Phe Gly Tyr Glu AlaGlu Ser Leu Met Ser Asn Leu Lys Val Lys Gly    50                  55                  60 Ser Phe Tyr Arg Asp LeuLys Arg Trp Val Gly Cys Asp Ser Ser Asn65                  70                  75                  80 Leu AspAla Tyr Leu Asp Arg Leu Lys Pro His Tyr Ser Val Arg Leu                85                  90                  95 Val Lys IleGly Ser Gly Leu Asn Glu Thr Val Ser Ile Gly Asn Phe            100                 105                 110 Gly Gly Thr ValLys Ser Glu Ala His Leu Pro Gly Leu Ile Ala Leu        115                 120                 125 Phe Ile Lys Ala ValIle Ser Cys Ala Glu Gly Ala Phe Ala Cys Thr    130                 135                 140 Cys Thr Gly Val Ile CysSer Val Pro Ala Asn Tyr Asp Ser Val Gln145                 150                 155                 160 Arg AsnPhe Thr Asp Gln Cys Val Ser Leu Ser Gly Tyr Gln Cys Val                165                 170                 175 Tyr Met IleAsn Glu Pro Ser Ala Ala Ala Leu Ser Ala Cys Asn Ser            180                 185                 190 Ile Gly Lys LysSer Ala Asn Leu Ala Val Tyr Asp Phe Gly Gly Gly        195                 200                 205 Thr Phe Asp Val SerIle Ile Ser Tyr Arg Asn Asn Thr Phe Val Val    210                 215                 220 Arg Ala Ser Gly Gly AspLeu Asn Leu Gly Gly Arg Asp Val Asp Arg225                 230                 235                 240 Ala PheLeu Thr His Leu Phe Ser Leu Thr Ser Leu Glu Pro Asp Leu                245                 250                 255 Thr Leu AspIle Ser Asn Leu Lys Glu Ser Leu Ser Lys Thr Asp Ala            260                 265                 270 Glu Ile Val TyrThr Leu Arg Gly Val Asp Gly Arg Lys Glu Asp Val        275                 280                 285 Arg Val Asn Lys AsnIle Leu Thr Ser Val Met Leu Pro Tyr Val Asn    290                 295                 300 Arg Thr Leu Lys Ile LeuGlu Ser Thr Leu Lys Ser Tyr Ala Lys Ser305                 310                 315                 320 Met AsnGlu Ser Ala Arg Val Lys Cys Asp Leu Val Leu Ile Gly Gly                325                 330                 335 Ser Ser TyrLeu Pro Gly Leu Ala Asp Val Leu Thr Lys His Gln Ser            340                 345                 350 Val Asp Arg IleLeu Arg Val Ser Asp Pro Arg Ala Ala Val Ala Val        355                 360                 365 Gly Cys Ala Leu TyrSer Ser Cys Leu Ser Gly Ser Gly Gly Leu Leu    370                 375                 380 Leu Ile Asp Cys Ala AlaHis Thr Val Ala Ile Ala Asp Arg Ser Cys385                 390                 395                 400 His GlnIle Ile Cys Ala Pro Ala Gly Ala Pro Ile Pro Phe Ser Gly                405                 410                 415 Ser Met ProLeu Tyr Leu Ala Arg Val Asn Lys Asn Ser Gln Arg Glu            420                 425                 430 Val Ala Val PheGlu Gly Glu Tyr Val Lys Cys Pro Lys Asn Arg Lys        435                 440                 445 Ile Cys Gly Ala AsnIle Arg Phe Phe Asp Ile Gly Val Thr Gly Asp    450                 455                 460 Ser Tyr Ala Pro Val ThrPhe Tyr Met Asp Phe Ser Ile Ser Ser Val465                 470                 475                 480 Gly AlaVal Ser Phe Val Val Arg Gly Pro Glu Gly Lys Gln Val Ser                485                 490                 495 Leu Thr GlyThr Pro Ala Tyr Asn Phe Ser Ser Val Ala Leu Gly Ser            500                 505                 510 Arg Ser Val ArgGlu Leu His Ile Ser Leu Asn Asn Lys Val Phe Leu        515                 520                 525 Gly Leu Leu Leu HisArg Lys Ala Asp Arg Arg Ile Leu Phe Thr Lys    530                 535                 540 Asp Glu Ala Ile Arg TyrAla Asp Ser Ile Asp Ile Ala Asp Val Leu545                 550                 555                 560 Lys GluTyr Lys Ser Tyr Ala Ala Ser Ala Leu Pro Pro Asp Glu Asp                565                 570                 575 Val Glu LeuLeu Leu Gly Lys Ser Val Gln Lys Val Leu Arg Gly Ser            580                 585                 590 Arg Leu Glu GluIle Pro Leu         595and a molecular weight from about 63 to about 67 kDa, preferably about65 kDa.

Another such DNA molecule (GLRaV-2 ORF4) includes nucleotides11277-12932 of SEQ. ID. No. 1 and codes for a putative grapevineleafroll virus heat shock 90 protein. This DNA molecule comprises anucleotide sequence corresponding to SEQ. ID. No. 10 as follows:ATGTCGAATT ACTCCTGGGA AAGTCTGTTC AAAAAGTTTT ACGGGGAAGC AGACTGGAAG   60AAATACCTCT CTAGGAGCAT AGCAGCACAC TCAAGTGAAA TTAAAACTCT ACCAGACATT  120CGATTGTACG GCGGTAGGGT TGTAAAGAAG TCCGAATTCG AATCAGCACT TCCTAATTCT  180TTTGAACAGG AATTAGGACT GTTCATACTG AGCGAACGGG AAGTGGGATG GAGCAAATTA  240TGCGGAATAA CGGTGGAAGA AGCAGCATAC GATCTTACGA ATCCCAAGGC TTATAAATTC  300ACTGCCGAGA CATGTAGCCC GGATGTAAAA GGTGAAGGAC AAAAATACTC TATGGAAGAC  360GTGATGAATT TCATGCGTTT ATCAAATCTG GATGTTAACG ACAAGATGCT GACGGAACAG  420TGTTGGTCGC TGTCCAATTC ATGCGGTGAA TTGATCAACC CAGACGACAA AGGGCGATTC  480GTGGCTCTCA CCTTTAAGGA CAGAGACACA GCTGATGACA CGGGTGCCGC CAACGTGGAA  540TGTCGCGTGG GCGACTATCT AGTTTACGCT ATGTCCCTGT TTGAGCAGAG GACCCAAAAA  600TCGCAGTCTG GCAACATCTC TCTGTACGAA AAGTACTGTG AATACATCAG GACCTACTTA  660GGGAGTACAG ACCTGTTCTT CACAGCGCCG GACAGGATTC CGTTACTTAC GGGCATCCTA  720TACGATTTTT GTAAGGAATA CAACGTTTTC TACTCGTCAT ATAAGAGAAA CGTCGATAAT  780TTCAGATTCT TCTTGGCGAA TTATATGCCT TTGATATCTG ACGTCTTTGT CTTCCAGTGG  840GTAAAACCCG CGCCGGATGT TCGGCTGCTT TTTGAGTTAA GTGCAGCGGA ACTAACGCTG  900GAGGTTCCCA CACTGAGTTT GATAGATTCT CAAGTTGTGG TAGGTCATAT CTTAAGATAC  960GTAGAATCCT ACACATCAGA TCCAGCCATC GACGCGTTAG AAGACAAACT GGAAGCGATA 1020CTGAAAAGTA GCAATCCCCG TCTATCGACA GCGCAACTAT GGGTTGGTTT CTTTTGTTAC 1080TATGGTGAGT TTCGTACGGC TCAAAGTAGA GTAGTGCAAA GACCAGGCGT ATACAAAACA 1140CCTGACTCAG TGGGTGGATT TGAAATAAAC ATGAAAGATG TTGAGAAATT CTTCGATAAA 1200CTTCAGAGAG AATTGCCTAA TGTATCTTTG CGGCGTCAGT TTAACGGAGC TAGAGCGCAT 1260GAGGCTTTCA AAATATTTAA AAACGGAAAT ATAAGTTTCA GACCTATATC GCGTTTAAAC 1320GTGCCTAGAG AGTTCTGGTA TCTGAACATA GACTACTTCA GGCACGCGAA TAGGTCCGGG 1380TTAACCGAAG AAGAAATACT CATCCTAAAC AACATAAGCG TTGATGTTAG GAAGTTATGC 1440GCTGAGAGAG CGTGCAATAC CCTACCTAGC GCGAAGCGCT TTAGTAAAAA TCATAAGAGT 1500AATATACAAT CATCACGCCA AGAGCGGAGG ATTAAAGACC CATTGGTAGT CCTGAAAGAC 1560ACTTTATATG AGTTCCAACA CAAGCGTGCC GGTTGGGGGT CTCGAAGCAC TCGAGACCTC 1620GGGAGTCGTG CTGACCACGC GAAAGGAAGC GGTTGA 1656

The heat shock 90 protein has an amino acid sequence corresponding toSEQ. ID. No. 11 as follows: Met Ser Asn Tyr Ser Trp Glu Ser Leu Phe LysLys Phe Tyr Gly Glu1               5                   10                  15 Ala Asp TrpLys Lys Tyr Leu Ser Arg Ser Ile Ala Ala His Ser Ser            20                  25                  30 Glu Ile Lys ThrLeu Pro Asp Ile Arg Leu Tyr Gly Gly Arg Val Val        35                  40                  45 Lys Lys Ser Glu PheGlu Ser Ala Leu Pro Asn Ser Phe Glu Gln Glu    50                  55                  60 Leu Gly Leu Phe Ile LeuSer Glu Arg Glu Val Gly Trp Ser Lys Leu65                  70                  75                  80 Cys GlyIle Thr Val Glu Glu Ala Ala Tyr Asp Leu Thr Asn Pro Lys                85                  90                  95 Ala Tyr LysPhe Thr Ala Glu Thr Cys Ser Pro Asp Val Lys Gly Glu            100                 105                 110 Gly Gln Lys TyrSer Met Glu Asp Val Met Asn Phe Met Arg Leu Ser        115                 120                 125 Asn Leu Asp Val AsnAsp Lys Met Leu Thr Glu Gln Cys Trp Ser Leu    130                 135                 140 Ser Asn Ser Cys Gly GluLeu Ile Asn Pro Asp Asp Lys Gly Arg Phe145                 150                 155                 160 Val AlaLeu Thr Phe Lys Asp Arg Asp Thr Ala Asp Asp Thr Gly Ala                165                 170                 175 Ala Asn ValGlu Cys Arg Val Gly Asp Tyr Leu Val Tyr Ala Met Ser            180                 185                 190 Leu Phe Glu GlnArg Thr Gln Lys Ser Gln Ser Gly Asn Ile Ser Leu        195                 200                 205 Tyr Glu Lys Tyr CysGlu Tyr Ile Arg Thr Tyr Leu Gly Ser Thr Asp    210                 215                 220 Leu Phe Phe Thr Ala ProAsp Arg Ile Pro Leu Leu Thr Gly Ile Leu225                 230                 235                 240 Tyr AspPhe Cys Lys Glu Tyr Asn Val Phe Tyr Ser Ser Tyr Lys Arg                245                 250                 255 Asn Val AspAsn Phe Arg Phe Phe Leu Ala Asn Tyr Met Pro Leu Ile            260                 265                 270 Ser Asp Val PheVal Phe Gln Trp Val Lys Pro Ala Pro Asp Val Arg        275                 280                 285 Leu Leu Phe Glu LeuSer Ala Ala Glu Leu Thr Leu Glu Val Pro Thr    290                 295                 300 Leu Ser Leu Ile Asp SerGln Val Val Val Gly His Ile Leu Arg Tyr305                 310                 315                 320 Val GluSer Tyr Thr Ser Asp Pro Ala Ile Asp Ala Leu Glu Asp Lys                325                 330                 335 Leu Glu AlaIle Leu Lys Ser Ser Asn Pro Arg Leu Ser Thr Ala Gln            340                 345                 350 Leu Trp Val GlyPhe Phe Cys Tyr Tyr Gly Glu Phe Arg Thr Ala Gln        355                 360                 365 Ser Arg Val Val GlnArg Pro Gly Val Tyr Lys Thr Pro Asp Ser Val    370                 375                 380 Gly Gly Phe Glu Ile AsnMet Lys Asp Val Glu Lys Phe Phe Asp Lys385                 390                 395                 400 Leu GlnArg Glu Leu Pro Asn Val Ser Leu Arg Arg Gln Phe Asn Gly                405                 410                 415 Ala Arg AlaHis Glu Ala Phe Lys Ile Phe Lys Asn Gly Asn Ile Ser            420                 425                 430 Phe Arg Pro IleSer Arg Leu Asn Val Pro Arg Glu Phe Trp Tyr Leu        435                 440                 445 Asn Ile Asp Tyr PheArg His Ala Asn Arg Ser Gly Leu Thr Glu Glu    450                 455                 460 Glu Ile Leu Ile Leu AsnAsn Ile Ser Val Asp Val Arg Lys Leu Cys465                 470                 475                 480 Ala GluArg Ala Cys Asn Thr Leu Pro Ser Ala Lys Arg Phe Ser Lys                485                 490                 495 Asn His LysSer Asn Ile Gln Ser Ser Arg Gln Glu Arg Arg Ile Lys            500                 505                 510 Asp Pro Leu ValVal Leu Lys Asp Thr Leu Tyr Glu Phe Gln His Lys        515                 520                 525 Arg Ala Gly Trp GlySer Arg Ser Thr Arg Asp Leu Gly Ser Arg Ala    530                 535                 540 Asp His Ala Lys Gly SerGly 545                 550and a molecular weight from about 61 to about 65 kDa, preferably about63 kDa.

Yet another DNA molecule of the present invention (GLRaV-2 ORF5)includes nucleotides 12844-13515 of SEQ. ID. No. 1 and codes for adiverged coat protein. This DNA molecule comprises a nucleotide sequencecorresponding to SEQ. ID. No. 12 as follows: ATGAGTTCCA ACACAAGCGTGCCGGTTGGG GGTCTCGAAG CACTCGAGAC CTCGGGAGTC  60 GTGCTGACCA CGCGAAAGGAAGCGGTTGAT AAGTTTTTTA ATGAACTAAA AAACGAAAAT 120 TACTCATCAG TTGACAGCAGCCGATTAAGC GATTCGGAAG TAAAAGAAGT GTTAGAGAAA 180 AGTAAAGAAA GTTTCAAAAGCGAACTGGCC TCCACTGACG AGCACTTCGT CTACCACATT 240 ATATTTTTCT TAATCCGATGTGCTAAGATA TCGACAAGTG AAAAGGTGAA GTACGTTGGT 300 AGTCATACGT ACGTGGTCGACGGAAAAACG TACACCGTTC TTGACGCTTG GGTATTCAAC 360 ATGATGAAAA GTCTCACGAAGAAGTACAAA CGAGTGAATG GTCTGCGTGC GTTCTGTTGC 420 GCGTGCGAAG ATCTATATCTAACCGTCGCA CCAATAATGT CAGAACGCTT TAAGACTAAA 480 GCCGTAGGGA TGAAAGGTTTGCCTGTTGGA AAGGAATACT TAGGCGCCGA CTTTCTTTCG 540 GGAACTAGCA AACTGATGAGCGATCACGAC AGGGCGGTCT CCATCGTTGC AGCGAAAAAC 600 GCTGTCGATC GTAGCGCTTTCACGGGTGGG GAGAGAAAGA TAGTTAGTTT GTATGATCTA 660 GGGAGGTACT AA 672

The diverged coat protein has an amino acid sequence corresponding toSEQ. ID. No. 13 as follows: Met Ser Ser Asn Thr Ser Val Pro Val Gly GlyLeu Glu Ala Leu Glu1               5                   10                  15 Thr Ser GlyVal Val Leu Thr Thr Arg Lys Glu Ala Val Asp Lys Phe            20                  25                  30 Phe Asn Glu LeuLys Asn Glu Asn Tyr Ser Ser Val Asp Ser Ser Arg        35                  40                  45 Leu Ser Asp Ser GluVal Lys Glu Val Leu Glu Lys Ser Lys Glu Ser    50                  55                  60 Phe Lys Ser Glu Leu AlaSer Thr Asp Glu His Phe Val Tyr His Ile65                  70                  75                  80 Ile PhePhe Leu Ile Arg Cys Ala Lys Ile Ser Thr Ser Glu Lys Val                85                  90                  95 Lys Tyr ValGly Ser His Thr Tyr Val Val Asp Gly Lys Thr Tyr Thr            100                 105                 110 Val Leu Asp AlaTrp Val Phe Asn Met Met Lys Ser Leu Thr Lys Lys        115                 120                 125 Tyr Lys Arg Val AsnGly Leu Arg Ala Phe Cys Cys Ala Cys Glu Asp    130                 135                 140 Leu Tyr Leu Thr Val AlaPro Ile Met Ser Glu Arg Phe Lys Thr Lys145                 150                 155                 160 Ala ValGly Met Lys Gly Leu Pro Val Gly Lys Glu Tyr Leu Gly Ala                165                 170                 175 Asp Phe LeuSer Gly Thr Ser Lys Leu Met Ser Asp His Asp Arg Ala            180                 185                 190 Val Ser Ile ValAla Ala Lys Asn Ala Val Asp Arg Ser Ala Phe Thr        195                 200                 205 Gly Gly Glu Arg LysIle Val Ser Leu Tyr Asp Leu Gly Arg Tyr    210                 215                 220and a molecular weight from about 23 to about 27 kDa, preferably about25 kDa.

Another such DNA molecule (GLRaV-2 ORF6) includes nucleotides13584-14180 of SEQ. ID. No. 1 and codes for a grapevine leafroll viruscoat protein. This DNA molecule comprises a nucleotide sequencecorresponding to SEQ. ID. No. 14 as follows: ATGGAGTTGA TGTCCGACAGCAACCTTAGC AACCTGGTGA TAACCGACGC CTCTAGTCTA  60 AATGGTGTCG ACAAGAAGCTTTTATCTGCT GAAGTTGAAA AAATGTTGGT GCAGAAAGGG 120 GCTCCTAACG AGGGTATAGAAGTGGTGTTC GGTCTACTCC TTTACGCACT CGCGGCAAGA 180 ACCACGTCTC CTAAGGTTCAGCGCGCAGAT TCAGACGTTA TATTTTCAAA TAGTTTCGGA 240 GAGAGGAATG TGGTAGTAACAGAGGGTGAC CTTAAGAAGG TACTCGACGG GTGTGCGCCT 300 CTCACTAGGT TCACTAATAAACTTAGAACG TTCGGTCGTA CTTTCACTGA GGCTTACGTT 360 GACTTTTGTA TCGCGTATAAGCACAAATTA CCCCAACTCA ACGCCGCGGC GGAATTGGGG 420 ATTCCAGCTG AAGATTCGTACTTAGCTGCA GATTTTCTGG GTACTTGCCC GAAGCTCTCT 480 GAATTACAGC AAAGTAGGAAGATGTTCGCG AGTATGTACG CTCTAAAAAC TGAAGGTGGA 540 GTGGTAAATA CACCAGTGAGCAATCTGCGT CAGCTAGGTA GAAGGGAAGT TATGTAA 597

The coat protein has an amino acid sequence corresponding to SEQ. ID.No. 15 as follows: Met Glu Leu Met Ser Asp Ser Asn Leu Ser Asn Leu ValIle Thr Asp 1               5                   10                  15Ala Ser Ser Leu Asn Gly Val Asp Lys Lys Leu Leu Ser Ala Glu Val            20                  25                  30 Glu Lys Met LeuVal Gln Lys Gly Ala Pro Asn Glu Gly Ile Glu Val        35                  40                  45 Val Phe Gly Leu LeuLeu Tyr Ala Leu Ala Ala Arg Thr Thr Ser Pro    50                  55                  60 Lys Val Gln Arg Ala AspSer Asp Val Ile Phe Ser Asn Ser Phe Gly65                  70                  75                  80 Glu ArgAsn Val Val Val Thr Glu Gly Asp Leu Lys Lys Val Leu Asp                85                  90                  95 Gly Cys AlaPro Leu Thr Arg Phe Thr Asn Lys Leu Arg Thr Phe Gly            100                 105                 110 Arg Thr Phe ThrGlu Ala Tyr Val Asp Phe Cys Ile Ala Tyr Lys His        115                 120                 125 Lys Leu Pro Gln LeuAsn Ala Ala Ala Glu Leu Gly Ile Pro Ala Glu    130                 135                 140 Asp Ser Tyr Leu Ala AlaAsp Phe Leu Gly Thr Cys Pro Lys Leu Ser145                 150                 155                 160 Glu LeuGln Gln Ser Arg Lys Met Phe Ala Ser Met Tyr Ala Leu Lys                165                 170                 175 Thr Glu GlyGly Val Val Asn Thr Pro Val Ser Asn Leu Arg Gln Leu            180                 185                 190 Gly Arg Arg GluVal Met         195and a molecular weight from about 20 to about 24 kDa, preferably about22 kDa.

Another such DNA molecule (GLRaV-2 ORF7) includes nucleotides14180-14665 of SEQ. ID. No. 1 and codes for a second undefined grapevineleafroll virus protein or polypeptide. This DNA molecule comprises anucleotide sequence corresponding to SEQ. ID. No. 16 as follows:ATGGAAGATT ACGAAGAAAA ATCCGAATCG CTCATACTGC TACGCACGAA TCTGAACACT  60ATGCTTTTAG TGGTCAAGTC CGATGCTAGT GTAGAGCTGC CTAAACTACT AATTTGCGGT 120TACTTACGAG TGTCAGGACG TGGGGAGGTG ACGTGTTGCA ACCGTGAGGA ATTAACAAGA 180GATTTTGAGG GCAATCATCA TACGGTGATC CGTTCTAGAA TCATACAATA TGACAGCGAG 240TCTGCTTTTG AGGAATTCAA CAACTCTGAT TGCGTAGTGA AGTTTTTCCT AGAGACTGGT 300AGTGTCTTTT GGTTTTTCCT TCGAAGTGAA ACCAAAGGTA GAGCGGTGCG ACATTTGCGC 360ACCTTCTTCG AAGCTAACAA TTTCTTCTTT GGATCGCATT GCGGTACCAT GGAGTATTGT 420TTGAAGCAGG TACTAACTGA AACTGAATCT ATAATCGATT CTTTTTGCGA AGAAAGAAAT 480CGTTAA 486

The second undefined grapevine leafroll virus protein or polypeptide hasa deduced amino acid sequence corresponding to SEQ. ID. No. 17 asfollows: Met Glu Asp Tyr Glu Glu Lys Ser Glu Ser Leu Ile Leu Leu Arg Thr1               5                   10                  15 Asn Leu AsnThr Met Leu Leu Val Val Lys Ser Asp Ala Ser Val Glu            20                  25                  30 Leu Pro Lys LeuLeu Ile Cys Gly Tyr Leu Arg Val Ser Gly Arg Gly        35                  40                  45 Glu Val Thr Cys CysAsn Arg Glu Glu Leu Thr Arg Asp Phe Glu Gly    50                  55                  60 Asn His His Thr Val IleArg Ser Arg Ile Ile Gln Tyr Asp Ser Glu65                  70                  75                  80 Ser AlaPhe Glu Glu Phe Asn Asn Ser Asp Cys Val Val Lys Phe Phe                85                  90                  95 Leu Glu ThrGly Ser Val Phe Trp Phe Phe Leu Arg Ser Glu Thr Lys            100                 105                 110 Gly Arg Ala ValArg His Leu Arg Thr Phe Phe Glu Ala Asn Asn Phe        115                 120                 125 Phe Phe Gly Ser HisCys Gly Thr Met Glu Tyr Cys Leu Lys Gln Val    130                 135                 140 Leu Thr Glu Thr Glu SerIle Ile Asp Ser Phe Cys Glu Glu Arg Asn145                 150                 155                 160 Argand a molecular weight from about 17 to about 21 kDa, preferably about19 kDa.

Yet another such DNA molecule (GLRaV-2 ORF8) includes nucleotides14667-15284 of SEQ. ID. No. 1 and codes for a third undefined grapevineleafroll virus protein or polypeptide. This DNA molecule comprises anucleotide sequence corresponding to SEQ. ID. No. 18 as follows:ATGAGGGTTA TAGTGTCTCC TTATGAAGCT GAAGACATTC TGAAAAGATC GACTGACATG  60TTACGAAACA TAGACAGTGG GGTCTTGAGC ACTAAAGAAT GTATCAAGGC ATTCTCGACG 120ATAACGCGAG ACCTACATTG TGCGAAGGCT TCCTACCAGT GGGGTGTTGA CACTGGGTTA 180TATCAGCGTA ATTGCGCTGA AAAACGTTTA ATTGACACGG TGGAGTCAAA CATACGGTTG 240GCTCAACCTC TCGTGCGTGA AAAAGTGGCG GTTCATTTTT GTAAGGATGA ACCAAAAGAG 300CTAGTAGCAT TCATCACGCG AAAGTACGTG GAACTCACGG GCGTGGGAGT GAGAGAAGCG 360GTGAAGAGGG AAATGCGCTG TCTTACCAAA ACAGTTTTAA ATAAAATGTC TTTGGAAATG 420GCGTTTTACA TGTCACCACG AGCGTGGAAA AACGCTGAAT GGTTAGAACT AAAATTTTCA 480CCTGTGAAAA TCTTTAGAGA TCTGCTATTA GACGTGGAAA CGCTCAACGA ATTGTGCGCC 540GAAGATGATG TTCACGTCGA CAAAGTAAAT GAGAATGGGG ACGAAAATCA CGACCTCGAA 600CTCCAAGACG AATGTTAA 618

The third undefined protein or polypeptide has a deduced amino acidsequence corresponding to SEQ. ID. No. 19 as follows: Met Arg Val IleVal Ser Pro Tyr Glu Ala Glu Asp Ile Leu Lys Arg1               5                   10                  15 Ser Thr AspMet Leu Arg Asn Ile Asp Ser Gly Val Leu Ser Thr Lys            20                  25                  30 Glu Cys Ile LysAla Phe Ser Thr Ile Thr Arg Asp Leu His Cys Ala        35                  40                  45 Lys Ala Ser Tyr GlnTrp Gly Val Asp Thr Gly Leu Tyr Gln Arg Asn    50                  55                  60 Cys Ala Glu Lys Arg LeuIle Asp Thr Val Glu Ser Asn Ile Arg Leu65                  70                  75                  80 Ala GlnPro Leu Val Arg Glu Lys Val Ala Val His Phe Cys Lys Asp                85                  90                  95 Glu Pro LysGlu Leu Val Ala Phe Ile Thr Arg Lys Tyr Val Glu Leu            100                 105                 110 Thr Gly Val GlyVal Arg Glu Ala Val Lys Arg Glu Met Arg Ser Leu        115                 120                 125 Thr Lys Thr Val LeuAsn Lys Met Ser Leu Glu Met Ala Phe Tyr Met    130                 135                 140 Ser Pro Arg Ala Trp LysAsn Ala Glu Trp Leu Glu Leu Lys Phe Ser145                 150                 155                 160 Pro ValLys Ile Phe Arg Asp Leu Leu Leu Asp Val Glu Thr Leu Asn                165                 170                 175 Glu Leu CysAla Glu Asp Asp Val His Val Asp Lys Val Asn Glu Asn            180                 185                 190 Gly Asp Glu AsnHis Asp Leu Glu Leu Gln Asp Glu Cys        195                 200                 205and a molecular weight from about 22 to about 26 kDa, preferably about24 kDa.

Another DNA molecule of the present invention (GLRaV-2 3′ UTR) includesnucleotides 15285-15500 of SEQ. ID. No. 1 and comprises a nucleotidesequence corresponding to SEQ. ID. No. 23 as follows: ACATTGGTTAAGTTTAACGA AAATGATTAG TAAATAATAA ATCGAACGTG GGTGTATCTA  60 CCTGACGTATCAACTTAAGC TGTTACTGAG TAATTAAACC AACAAGTGTT GGTGTTAATGT 120 GTATGTTGATGTAGAGAAAA ATCCGTTTGT AGAACGGTGT TTTTCTCTTC TTTATTTTTA 180 AAAAAAAAATAAAAAAAAAA AAAAAAAAGC GGCCGC 216

Also encompassed by the present invention are fragments of the DNAmolecules of the present invention. Suitable fragments capable ofimparting grapevine leafroll resistance to grape plants are constructedby using appropriate restriction sites, revealed by inspection of theDNA molecule's sequence, to: (i) insert an interposon (Felley et al.,“Interposon Mutagenesis of Soil and Water Bacteria: a Family of DNAFragments Designed for in vitro Insertion Mutagenesis of Gram-negativeBacteria,” Gene, 52: 147-15 (1987), which is hereby incorporated byreference) such that truncated forms of the grapevine leafroll viruscoat polypeptide or protein, that lack various amounts of theC-terminus, can be produced or (ii) delete various internal portions ofthe protein. Alternatively, the sequence can be used to amplify anyportion of the coding region, such that it can be cloned into a vectorsupplying both transcription and translation start signals.

Suitable DNA molecules are those that hybridize to a DNA moleculecomprising a nucleotide sequence of at least 15 continuous bases of SEQ.ID. No. 1 under stringent conditions characterized by a hybridizationbuffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of37° C. and remaining bound when subject to washing with SSC buffer at37° C.; and preferably in a hybridization buffer comprising 20%formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42° C. andremaining bound when subject to washing at 42° C. with 0.2×SSC buffer at42° C.

Variants may also (or alternatively) be modified by, for example, thedeletion or addition of nucleotides that have minimal influence on theproperties, secondary structure and hydropathic nature of the encodedpolypeptide. For example, the nucleotides encoding a polypeptide may beconjugated to a signal (or leader) sequence at the N-terminal end of theprotein which co-translationally or post-translationally directstransfer of the protein. The nucleotide sequence may also be altered sothat the encoded polypeptide is conjugated to a linker or other sequencefor ease of synthesis, purification, or identification of thepolypeptide.

The protein or polypeptide of the present invention is preferablyproduced in purified form (preferably, at least about 80%, morepreferably 90%, pure), by conventional techniques. Typically, theprotein or polypeptide of the present invention is isolated by lysingand sonication. After washing, the lysate pellet is resuspended inbuffer containing Tris-HCl. During dialysis, a precipitate forms fromthis protein solution. The solution is centrifuged, and the pellet iswashed and resuspended in the buffer containing Tris-HCl. Proteins areresolved by electrophoresis through an SDS 12% polyacrylamide gel.

The DNA molecule encoding the grapevine leafroll virus (type 2) proteinor polypeptide of the present invention can be incorporated in cellsusing conventional recombinant DNA technology. Generally, this involvesinserting the DNA molecule into an expression system to which the DNAmolecule is heterologous (i.e. not normally present). The heterologousDNA molecule is inserted into the expression system or vector in propersense orientation and correct reading frame. The vector contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference, describes the production of expression systems in the formof recombinant plasmids using restriction enzyme cleavage and ligationwith DNA ligase. These recombinant plasmids are then introduced by meansof transformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vacciniavirus. Recombinant viruses can be generated by transfection of plasmidsinto cells infected with virus.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/−or KS +/−(see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif., which is hereby incorporated byreference), pQE, pIH821, pGEX, pET series (see Studier et. al., “Use ofT7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene ExpressionTechnology, vol. 185 (1990), which is hereby incorporated by reference),and any derivatives thereof. Recombinant molecules can be introducedinto cells via transformation, transduction, conjugation, mobilization,or electroporation. The DNA sequences are cloned into the vector usingstandard cloning procedures in the art, as described by Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, ColdSprings Harbor, N.Y. (1982), which is hereby incorporated by reference.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria ortransformed via particle bombardment (i.e. biolistics). The expressionelements of these vectors vary in their strength and specificities.Depending upon the host-vector system utilized, any one of a number ofsuitable transcription and translation elements can be used.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation).

Transcription of DNA is dependent upon the presence of a promoter whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eucaryotic promotersdiffer from those of procaryotic promoters. Furthermore, eucaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a procaryotic system, and, further, procaryoticpromoters are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presenceof the proper procaryotic signals which differ from those of eucaryotes.Efficient translation of mRNA in procaryotes requires a ribosome bindingsite called the Shine-Dalgamo (“SD”) sequence on the mRNA. This sequenceis a short nucleotide sequence of mRNA that is located before the startcodon, usually AUG, which encodes the amino-terminal methionine of theprotein. The SD sequences are complementary to the 3′-end of the 16SrRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomesby duplexing with the rRNA to allow correct positioning of the ribosome.For a review on maximizing gene expression, see Roberts and Lauer,Methods in Enzymology, 68: 473 (1979), which is hereby incorporated byreference.

Promoters vary in their “strength” (i.e. their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operons, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ tothe initiation codon (“ATG”) to provide a ribosome binding site. Thus,any SD-ATG combination that can be utilized by host cell ribosomes maybe employed. Such combinations include but are not limited to the SD-ATGcombination from the cro gene or the N gene of coliphage lambda, or fromthe E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATGcombination produced by recombinant DNA or other techniques involvingincorporation of synthetic nucleotides may be used.

Once the isolated DNA molecules encoding the various grapevine leafrollvirus (type 2) proteins or polypeptides, as described above, have beencloned into an expression system, they are ready to be incorporated intoa host cell. Such incorporation can be carried out by the various formsof transformation noted above, depending upon the vector/host cellsystem. Suitable host cells include, but are not limited to, bacteria,virus, yeast, mammalian cells, insect, plant, and the like.

The present invention also relates to RNA molecules which encode thevarious grapevine leafroll virus (type 2) proteins or polypeptidesdescribed above. The transcripts can be synthesized using the host cellsof the present invention by any of the conventional techniques. The mRNAcan be translated either in vitro or in vivo. Cell-free systemstypically include wheat-germ or reticulocyte extracts. In vivotranslation can be effected, for example, by microinjection into frogoocytes.

One aspect of the present invention involves using one or more of theabove DNA molecules encoding the various proteins or polypeptides of agrapevine leafroll virus (type 2) to transform grape plants in order toimpart grapevine leafroll resistance to the plants. The mechanism bywhich resistance is imparted is not known. In one hypotheticalmechanism, the transformed plant can express a protein or polypeptide ofgrapevine leafroll virus (type 2), and, when the transformed plant isinoculated by a grapevine leafroll virus, such as GLRaV-1, GLRaV-2,GLRav-3, GLRaV-4, GLRaV-5, or GLRaV-6, or combinations of these, theexpressed protein or polypeptide prevents translation of the viral DNA.

In this aspect of the present invention the subject DNA moleculeincorporated in the plant can be constitutively expressed.Alternatively, expression can be regulated by a promoter which isactivated by the presence of grapevine leafroll virus. Suitablepromoters for these purposes include those from genes expressed inresponse to grapevine leafroll virus infiltration.

The isolated DNA molecules of the present invention can be utilized toimpart grapevine leafroll virus resistance for a wide variety ofgrapevine plants. The DNA molecules are particularly well suited toimparting resistance to Vitis scion or rootstock cultivars. Scioncultivars which can be protected include those commonly referred to asTable or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, AutumnBlack, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie,Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, CampbellEarly, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight,Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor,Exotic, Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay,Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, JulyMuscat, Khandahar, Katta, Kourgane, Kishmishi, Loose Perlette, Malaga,Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New YorkMuscat, Niabell, Niagara, Olivette blanche, Ontario, Pierce, Queen, RedMalaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca,Suavis (IP 365), Thompson seedless, and Thomuscat. They also includethose used in wine production, such as Aleatico, Alicante Bouschet,Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc,Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay, Chasselasdore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling,Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint,Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, GreenVeltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco deSalamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier,Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc, MuscatOttonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Lampia,Orange Muscat, Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah,Peverella, Pinot noir, Pinot Saint-George, Primitive di Gioa, RedVeltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby Cabernet,Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc,Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110,Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson,Sylvaner, Tannat, Teroldico, Tinta Madeira, Tinto cao, Touriga,Traminer, Trebbiano Toscano, Trousseau, Valdepenas, Viognier,Walschriesling, White Riesling, and Zinfandel. Rootstock cultivars whichcan be protected include Couderc 1202, Couderc 1613, Couderc 1616,Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A×R #1),Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & deGrasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (SO₄), Paulsen775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, RipariaGloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitisrupestris Constantia, Vitis california, and Vitis girdiana.

There exists an extensive similarity in the hsp70-related sequenceregions of GLRaV-2 and other closteroviruses, such as tristeza virus andbeet yellows virus. Consequently, the GLRaV-2 hsp70-related gene canalso be used to produce transgenic plants or cultivars other than grape,such as citrus or sugar beet, which are resistant to closterovirusesother than grapevine leafroll, such as tristeza virus and beet yellowsvirus.

Suitable citrus cultivars include lemon, lime, orange, grapefruit,pineapple, tangerine, and the like, such as Joppa, Maltaise Ovale,Parson (Parson Brown), Pera, Pineapple, Queen, Shamouti, Valencia,Tenerife, Imperial Doblefina, Washington Sanguine, Moro, SanguinelloMoscato, Spanish Sanguinelli, Tarocco, Atwood, Australian, Bahia,Baiana, Cram, Dalmau, Eddy, Fisher, Frost Washington, Gillette,LengNavelina, Washington, Satsuma Mandarin, Dancy, Robinson, Ponkan,Duncan, Marsh, Pink Marsh, Ruby Red, Red Seedless, Smooth Seville,Orlando Tangelo, Eureka, Lisbon, Meyer Lemon, Rough Lemon, Sour Orange,Persian Lime, West Indian Lime, Bearss, Sweet Lime, Troyer Citrange, andCitrus Trifoliata. Each of these citrus cultivars is suitable forproducing transgenic citrus plants resistant to tristeza virus.

The economically important species of sugar beet is Beta vulgaris L.,which has four important cultivar types: sugar beet, table beet, fodderbeet, and Swiss chard. Each of these beet cultivars is suitable forproducing transgenic beet plants resistant to beet yellows virus, asdescribed above.

Because GLRaV-2 has been known to infect tobacco plants (e.g., Nicotianabenthamiana), it is also desirable to produce transgenic tobacco plantswhich are resistant to grapevine leafroll viruses, such as GLRaV-2.

Plant tissue suitable for transformation include leaf tissue, roottissue, meristems, zygotic and somatic embryos, and anthers. It isparticularly preferred to utilize embryos obtained from anther cultures.

The expression system of the present invention can be used to transformvirtually any plant tissue under suitable conditions. Tissue cellstransformed in accordance with the present invention can be grown invitro in a suitable medium to impart grapevine leafroll virusresistance. Transformed cells can be regenerated into whole plants suchthat the protein or polypeptide imparts resistance to grapevine leafrollvirus in the intact transgenic plants. In either case, the plant cellstransformed with the recombinant DNA expression system of the presentinvention are grown and caused to express that DNA molecule to produceone of the above-described grapevine leafroll virus proteins orpolypeptides and, thus, to impart grapevine leafroll virus resistance.

In producing transgenic plants, the DNA construct in a vector describedabove can be microinjected directly into plant cells by use ofmicropipettes to transfer mechanically the recombinant DNA. Crossway,Mol. Gen. Genetics, 202: 179-85 (1985), which is hereby incorporated byreference. The genetic material may also be transferred into the plantcell using polyethylene glycol. Krens, et al., Nature, 296: 72-74(1982), which is hereby incorporated by reference.

One technique of transforming plants with the DNA molecules inaccordance with the present invention is by contacting the tissue ofsuch plants with an inoculum of a bacteria transformed with a vectorcomprising a gene in accordance with the present invention which impartsgrapevine leafroll resistance. Generally, this procedure involvesinoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25-28° C.

Bacteria from the genus Agrobacterium can be utilized to transform plantcells. Suitable species of such bacterium include Agrobacteriumtumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens(e.g., strains C58, LBA4404, or EHA 105) is particularly useful due toits well-known ability to transform plants.

Heterologous genetic sequences can be introduced into appropriate plantcells, by means of the Ti plasmid of A. tumefaciens or the R1 plasmid ofA. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells oninfection by Agrobacterium and is stably integrated into the plantgenome. J. Schell, Science. 237: 1176-83 (1987), which is herebyincorporated by reference.

After transformation, the transformed plant cells must be regenerated.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III(1986), which are hereby incorporated by reference.

It is known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to, all major species ofsugarcane, sugar beets, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining explants is first provided. Callus tissue is formed andshoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these threevariables are controlled, then regeneration is usually reproducible andrepeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure so that theDNA construct is present in the resulting plants. Alternatively,transgenic seeds are recovered from the transgenic plants. These seedscan then be planted in the soil and cultivated using conventionalprocedures to produce transgenic plants.

Another approach to transforming plant cells with a gene which impartsresistance to pathogens is particle bombardment (also known as biolistictransformation) of the host cell. This can be accomplished in one ofseveral ways. The first involves propelling inert or biologically activeparticles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and inEmerschad et al., “Somatic Embryogenesis and Plant Development fromImmature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” PlantCell Reports, 14: 6-12 (1995) (“Emerschad (1995)”), which are herebyincorporated by reference. Generally, this procedure involves propellinginert or biologically active particles at the cells under conditionseffective to penetrate the outer surface of the cell and to beincorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Once a grape plant tissue, citrus plant tissue, beet plant tissue, ortobacco plant tissue is transformed in accordance with the presentinvention, the transformed tissue is regenerated to form a transgenicplant. Generally, regeneration is accomplished by culturing transformedtissue on medium containing the appropriate growth regulators andnutrients to allow for the initiation of shoot meristems. Appropriateantibiotics are added to the regeneration medium to inhibit the growthof Agrobacterium and to select for the development of transformed cells.Following shoot initiation, shoots are allowed to develop tissue cultureand are screened for marker gene activity.

The DNA molecules of the present invention can be made capable oftranscription to a messenger RNA, which, although encoding for agrapevine leafroll virus (type 2) protein or polypeptide, does nottranslate to the protein. This is known as RNA-mediated resistance. Whena Vitis scion or rootstock cultivar, or a citrus, beet, or tobaccocultivar, is transformed with such a DNA molecule, the DNA molecule canbe transcribed under conditions effective to maintain the messenger RNAin the plant cell at low level density readings. Density readings ofbetween 15 and 50 using a Hewlet ScanJet and Image Analysis Program arepreferred.

A portion of one or more DNA molecules of the present invention as wellas other DNA molecules can be used in a transgenic grape plant, citrusplant, beet plant, or tobacco plant in accordance with U.S. patentapplication Ser. No. 09/025,635, which is hereby incorporated herein byreference.

The grapevine leafroll virus (type 2) protein or polypeptide of thepresent invention can also be used to raise antibodies or bindingportions thereof or probes. The antibodies can be monoclonal orpolyclonal.

Monoclonal antibody production may be effected by techniques which arewell-known in the art. Basically, the process involves first obtainingimmune cells (lymphocytes) from the spleen of a mammal (e.g., mouse)which has been previously immunized with the antigen of interest eitherin vivo or in vitro. The antibody-secreting lymphocytes are then fusedwith (mouse) myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. The resulting fused cells, orhybridomas, are cultured, and the resulting colonies screened for theproduction of the desired monoclonal antibodies. Colonies producing suchantibodies are cloned, and grown either in vivo or in vitro to producelarge quantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, Nature, 256: 495 (1975), which is hereby incorporated byreference.

Mammalian lymphocytes are immunized by in vivo immunization of theanimal (e.g., a mouse) with the protein or polypeptide of the presentinvention. Such immunizations are repeated as necessary at intervals ofup to several weeks to obtain a sufficient titer of antibodies.Following the last antigen boost, the animals are sacrificed and spleencells removed.

Fusion with mammalian myeloma cells or other fusion partners capable ofreplicating indefinitely in cell culture is effected by standard andwell-known techniques, for example, by using polyethylene glycol (“PEG”)or other fusing agents. (See Milstein and Kohler, Eur. J. Immunol., 6:511 (1976), which is hereby incorporated by reference.) This immortalcell line, which is preferably murine, but may also be derived fromcells of other mammalian species, including but not limited to rats andhumans, is selected to be deficient in enzymes necessary for theutilization of certain nutrients, to be capable of rapid growth, and tohave good fusion capability. Many such cell lines are known to thoseskilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpolypeptide of the present invention subcutaneously to New Zealand whiterabbits which have first been bled to obtain pre-immune serum. Theantigens can be injected at a total volume of 100 μl per site at sixdifferent sites. Each injected material will contain syntheticsurfactant adjuvant pluronic polyols, or pulverized acrylamide gelcontaining the protein or polypeptide after SDS-polyacrylamide gelelectrophoresis. The rabbits are then bled two weeks after the firstinjection and periodically boosted with the same antigen three timesevery six weeks. A sample of serum is then collected 10 days after eachboost. Polyclonal antibodies are then recovered from the serum byaffinity chromatography using the corresponding antigen to capture theantibody. Ultimately, the rabbits are euthenized with pentobarbital 150mg/Kg IV. This and other procedures for raising polyclonal antibodiesare disclosed in Harlow et. al., editors, Antibodies: A LaboratoryManual (1988), which is hereby incorporated by reference.

In addition to utilizing whole antibodies, binding portions of suchantibodies can be used. Such binding portions include Fab fragments,F(ab′)₂ fragments, and Fv fragments. These antibody fragments can bemade by conventional procedures, such as proteolytic fragmentationprocedures, as described in Goding, Monoclonal Antibodies: Principlesand Practice, New York:Academic Press, pp. 98-118 (1983), which ishereby incorporated by reference.

The present invention also relates to probes found either in nature orprepared synthetically by recombinant DNA procedures or other biologicalprocedures. Suitable probes are molecules which bind to grapevineleafroll (type 2) viral antigens identified by the monoclonal antibodiesof the present invention. Such probes can be, for example, proteins,peptides, lectins, or nucleic acid probes.

The antibodies or binding portions thereof or probes can be administeredto grapevine leafroll virus infected scion cultivars or rootstockcultivars. Alternatively, at least the binding portions of theseantibodies can be sequenced, and the encoding DNA synthesized. Theencoding DNA molecule can be used to transform plants together with apromoter which causes expression of the encoded antibody when the plantis infected by grapevine leafroll virus. In either case, the antibody orbinding portion thereof or probe will bind to the virus and help preventthe usual leafroll response.

Antibodies raised against the GLRaV-2 proteins or polypeptides of thepresent invention or binding portions of these antibodies can beutilized in a method for detection of grapevine leafroll virus in asample of tissue, such as tissue (e.g., scion or rootstock) from a grapeplant or tobacco plant. Antibodies or binding portions thereof suitablefor use in the detection method include those raised against a helicase,a methyltransferase, a papain-like protease, an RNA-dependent RNApolymerase, a heat shock 70 protein, a heat shock 90 protein, a coatprotein, a diverged coat protein, or other proteins or polypeptides inaccordance with the present invention. Any reaction of the sample withthe antibody is detected using an assay system which indicates thepresence of grapevine leafroll virus in the sample. A variety of assaysystems can be employed, such as enzyme-linked immunosorbent assays,radioimmunoassays, gel diffusion precipitin reaction assays,immunodiffusion assays, agglutination assays, fluorescent immunoassays,protein A immunoassays, or immunoelectrophoresis assays.

Alternatively, grapevine leafroll virus can be detected in such a sampleusing a nucleotide sequence of the DNA molecule, or a fragment thereof,encoding for a protein or polypeptide of the present invention. Thenucleotide sequence is provided as a probe in a nucleic acidhybridization assay or a gene amplification detection procedure (e.g.,using a polymerase chain reaction procedure). The nucleic acid probes ofthe present invention may be used in any nucleic acid hybridizationassay system known in the art, including, but not limited to, Southernblots (Southern, E. M., “Detection of Specific Sequences Among DNAFragments Separated by Gel Electrophoresis,” J. Mol. Biol., 98: 503-17(1975), which is hereby incorporated by reference), Northern blots(Thomas, P. S., “Hybridization of Denatured RNA and Small DNA FragmentsTransferred to Nitrocellulose,” Proc. Nat'l Acad. Sci. USA, 77: 5201-05(1980), which is hereby incorporated by reference), and Colony blots(Grunstein, M., et al., “Colony Hybridization: A Method for theIsolation of Cloned cDNAs that Contain a Specific Gene,” Proc. Nat'lAcad. Sci. USA, 72: 3961-65 (1975), which is hereby incorporated byreference). Alternatively, the probes can be used in a geneamplification detection procedure (e.g., a polymerase chain reaction).Erlich, H. A., et. al., “Recent Advances in the Polymerase ChainReaction,” Science 252: 1643-51 (1991), which is hereby incorporated byreference. Any reaction with the probe is detected so that the presenceof a grapevine leafroll virus in the sample is indicated. Such detectionis facilitated by providing the probe of the present invention with alabel. Suitable labels include a radioactive compound, a fluorescentcompound, a chemiluminescent compound, an enzymatic compound, or otherequivalent nucleic acid labels.

Depending upon the desired scope of detection, it is possible to utilizeprobes having nucleotide sequences that correspond with conserved orvariable regions of the ORF or UTR. For example, to distinguish agrapevine leafroll virus from other related viruses (e.g., otherclosteroviruses), it is desirable to use probes which contain nucleotidesequences that correspond to sequences more highly conserved among allgrapevine leafroll viruses. Also, to distinguish between differentgrapevine leafroll viruses (i.e., GLRaV-2 from GLRaV-1, GLRaV-3,GLRaV-4, GLRaV-5, and GLRaV-6), it is desirable to utilize probescontaining nucleotide sequences that correspond to sequences less highlyconserved among the different grapevine leafroll viruses.

Nucleic acid (DNA or RNA) probes of the present invention will hybridizeto complementary GLRaV-2 nucleic acid under stringent conditions.Generally, stringent conditions are selected to be about 50° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. The T_(m) is dependent upon thesolution conditions and the base composition of the probe, and may becalculated using the following equation: $\begin{matrix}{T_{m} = {{79.8{{^\circ}C}} + \left( {18.5 \times {{Log}\left\lbrack {{Na} +} \right\rbrack}} \right) + \left( {58.4{{^\circ}C} \times {\%\left\lbrack {G + C} \right\rbrack}} \right) -}} \\{\left( {{820/\#}{bp}\quad{in}\quad{duplex}} \right) - \left( {0.5 \times \%\quad{formamide}} \right)}\end{matrix}$Nonspecific binding may also be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein-containing solutions, addition of heterologous RNA, DNA, and SDSto the hybridization buffer, and treatment with RNase. Wash conditionsare typically performed at or below stringency. Generally, suitablestringent conditions for nucleic acid hybridization assays or geneamplification detection procedures are asas set forth above. More orless stringent conditions may also be selected.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1 Northern Hybridization

Specificity of the selected clones was confirmed by Northernhybridization. Northern hybridization was performed afterelectrophoresis of the dsRNA of GLRaV-2 in 1% agarose non-denaturingcondition gel. The agarose gel was denatured by soaking in 50 mM NaOHcontaining 0.4 M NaCl for 30 min, and then neutralized with 0.1 MTris-HCl (PH7.5) containing 0.5 M NaCl for another 30 min. RNA wassandwich blotted overnight onto Genescreen™ plus membrane (Dupont NENResearch Product) in 10×SSC buffer and hybridized as described by themanufacturer's instructions (DuPont, NEN).

Example 2 Sequencing and Computer Assisted Nucleotide and Amino AcidSequence Analysis

DNA inserts were sequenced in pBluescript SK+ by using T3 and T7universal primers for the terminal region sequence and additionaloligonucleotide primers designed according to the known sequence for theinternal region sequence. Purification of plasmid DNA was performed by amodified mini alkaline-lysis/PEG precipitation procedure described bythe manufacturer (Applied Biosystems, Inc.). Nucleotide sequencing wasperformed on both strands of cDNA by using ABI TaqDyeDeoxy TerminatorCycle Sequencing Kit (Applied Biosystems, Inc.). Automatic sequencingwas performed on an ABI373 Automated Sequencer (Applied Biosynstems,Inc.) at Cornell University, Geneva, N.Y.

The nucleotide sequences of GLRaV-2 were assembled and analyzed with theprograms of EditSeq and SeqMan, respectively, of DNASTAR package(Madison, Wis.). Amino acid sequences deduced from nucleotide sequencesand its encoding open reading frames were conducted using the MapDrawprogram. Multiple alignments of amino acid sequences, identification ofconsensus amino acid sequences, and generation of phylogenetic treeswere performed using the Clustal method in the MegAlign program. Thenucleotide and amino acid sequences of other closteroviruses wereobtained with the Entrez Program; and sequence comparisons withnonredundant databases were searched with the Blast Program from theNational Center for Biotechnology Information.

Example 3 Isolation of dsRNA

Several vines of GLRaV-2 infected Vitis vinifera cv Pinot Noir thatoriginated from a central New York vineyard served as the source fordsRNA isolation and cDNA cloning. dsRNA was extracted from phloem tissueof infected grapevines according to the method described by Hu et al.,“Characterization of Closterovirus-Like Particles Associated withGrapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), whichis hereby incorporated by reference. Purification of the high molecularweight dsRNA (ca 15 kb) was carried out by electrophoretic separation ofthe total dsRNA on a 0.7% low melting point agarose gel and extractionby phenol/chloroform following the method described by Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Sping HarborLaboratory Press, New York (1989), which is hereby incorporated byreference. Concentration of dsRNA was estimated with UV fluorescentdensity of an ethidium bromide stained dsRNA band in comparison with aknown concentration of DNA marker.

Example 4 cDNA Synthesis and Cloning

cDNA synthesis was performed following the method initially described byJelkmann et al., “Cloning of Four Plant Viruses From Small Quantities ofDouble-Stranded RNA,” Phytopathology 79: 1250-53 (1989) and modified byLing et al., “The Coat Protein Gene of Grapevine Leafroll AssociatedClosterovirus-3: Cloning, Nucleotide Sequencing and Expression inTransgenic Plants,” Arch. Virology 142: 1101-16 (1997), both of whichare hereby incorporated by reference. About 100 ng of high molecularweight dsRNA purified from low melting agarose gel was denatured in 20mM methylmercuric hydroxide and incubated at room temperature for 10 minwith 350 ng of random primers. First strand cDNA was synthesized byusing avian myeloblastosis virus (AMV) reverse transcriptase. Secondstrand cDNA was obtained by using RNase H and E. coli DNA polymerase I.Double-stranded cDNA was blunt ended with T4 DNA polymerase and ligatedwith EcoR I adapters. The cDNA, which had EcoR I adapters at the ends,was activated by kinase reaction and ligated into Lambda ZAP II/EcoR Iprepared arms following the manufacturer's instruction (Stratagene). Therecombinant DNA was then packaged in vitro to Gigapack® II packagingextract (Stratagene). The packaged phage particles were amplified andtitered according to the manufacturer's instruction.

Two kinds of probes were used to identify GLRaV-2 specific clones fromthe library. One type was prepared from the synthesized cDNA that wasamplified by PCR after ligation to the specific EcoR I Uni-Amp™ adapters(Clontech); and the other type was DNA inserts or PCR products fromalready sequenced clones. Clones from the cDNA library were selected bycolony-lifting hybridization onto the colony/plaque Screen membrane (NENResearch Product) with the probe described above. The probe was preparedby labeling with ³²P [α-dATP] using Klenow fragment of E. coli DNApolymerase I. Prehybridization, hybridization, and washing steps werecarried out at 65° C. according to the manufacturer's instruction(Dupont, NEN Research Product). Selected plaques were converted torecombinant pBluescript by in vivo excision method according to themanufacturer's instruction (Stratagene).

To obtain clones representing the extreme 3′-terminus of GLRaV-2, dsRNAwas polyadenylated by yeast poly(A) polymerase. Using poly(A)-taileddsRNA as template, cDNA was amplified by RT-PCR with oligo(dT)18 and aspecific primer, CP-1/T7R, which is derived from the clone CP-1 and hasa nucleotide sequence according to SEQ. ID. No. 20 as follows:TGCTGGAGCT TGAGGTTCTG C 21The resulting PCR product (3′-PCR) was cloned into a TA vector(Invitrogen) and sequenced.

As shown in FIG. 1A, a high molecular weight dsRNA of ca. 15 kb wasconsistently identified from GLRaV-2 infected grapevines, but not fromhealthy vines. In addition, several low molecular weight dsRNAs werealso detected from infected tissue. The yield of dsRNA of GLRaV-2 wasestimated between 5-10 ng/15 g phloem tissue, which was much lower thanthat of GLRaV-3 (Hu et al., “Characterization of Closterovirus-LikeParticles Associated with Grapevine Leafroll Disease,” J. Phytopathology128: 1-14 (1990), which is hereby incorporated by reference). Only thehigh molecular weight dsRNA that was purified from low melting pointagarose gel was used for cDNA synthesis, cloning and establishment ofthe Lambda/ZAP II cDNA library.

Two kinds of probes were used for screening the cDNA library. Theinitial clones were identified by hybridization with Uni-Amp™PCR-amplified cDNA as probes. The specificity of these clones (e.g.,TC-1) ranging from 200 to 1,800 bp in size was confirmed by Northernhybridization to dsRNA of GLRaV-2 as shown in FIG. 1B. Additionally,over 40 different clones ranging form 800 to 7,500 bp in size wereidentified following hybridization with the probes generated fromGLRaV-2 specific cDNA clones or from PCR products. Over 40 clones werethen sequenced on the both strands (FIG. 2).

Example 5 Expression of the Coat Protein in E. coli and Immunoblotting

To determine that ORF6 was the coat protein gene of GLRaV-2, thecomplete ORF6 DNA molecule was subcloned from a PCR product and insertedinto the fusion protein expression vector pMAL-C2 (New England Biolabs,Inc.). The specific primers used for the PCR reaction were CP-96F andCP-96R, in which an EcoR I or BamH I site was included to facilitatecloning. CP-96F was designed to include the start codon of the CP andcomprises a nucleotide sequence according to SEQ. ID. NO. 21 as follows:

-   CGGAATTCAC CATGGAGTTG ATGTCCGACA G 31

CP-96R was 66 nucleotides downstream of the stop codon of the CP andcomprises the nucleotide sequence corresponding to SEQ. ID. No.22 asfollows: AGCGGATCCA TGGCAGATTC GTGCGTAGCA GTA 33The coat protein was expressed as a fusion protein with maltose bindingprotein (MBP) of E. coli under the control of a “tac” promoter andsuppressed by the “lac” repressor. The MBP-CP fusion protein was inducedby adding 0.3 mM isopropyl-β-D-thio-gloactopyranoside (IPTG) andpurified by a one step affinity column according to the manufacturer'sinstruction (New England, Biolabs, Inc). The MBP-CP fusion protein orthe coat protein cleaved from the fusion protein was tested to reactwith specific antiserum of GLRaV-2 (kindly provided by Dr. Charles Greifof INRA, Colmar, France) on Western blot according to the methoddescribed by Hu et al., “Characterization of Closterovirus-LikeParticles Associated with Grapevine Leafroll Disease,” J. Phytopathology128: 1-14 (1990), which is hereby incorporated by reference. Incontrast, the non-recombinant plasmids or uninduced cells did not reactto the antiserum of GLRaV-2.

Example 6 Sequence Analysis and Genome Organization of GLRaV-2

A total of 15,500 bp of the RNA genome of GLRaV-2 was sequenced anddeposited in GenBank (accession number AF039204). About 85% of the totalRNA genome was revealed from at least two different clones. The sequencein the coat protein gene region was determined and confirmed fromseveral different overlapping clones. The genome organization ofGLRaV-2, shown in FIG. 2, includes nine open reading frames (e.g.,ORF1a, 1b-8).

ORF1a and ORF1b: Analysis of the amino acid sequence of the N-terminalportion of GLRaV-2 ORF 1 a encoded product revealed two putativepapain-like protease domains, which showed significant similarity to thepapain-like leader protease of BYV (Agranovsky et al., “Beet YellowsClosterovirus: Complete Genome Structure and Identification of aPapain-like Thiol Protease,” Virology 198: 311-24 (1994), which ishereby incorporated by reference). Thus, it allowed prediction of thecatalytic cysteine and histidine residues for the putative GLRaV-2protease. Upon alignment of the sequence of the papain-like protease ofBYV with that of GLRaV-2, the cleavage site at residues Gly-Gly (aminoacid 588-589) of BYV aligned with the corresponding alanine-glycine(Ala-Gly) and Gly-Gly dipeptide of GLRaV-2 (FIG. 3A). Cleavage at thissite would result in a leader protein and a 234 kDa (2090 amino acid)C-terminal fragment consisting of MT and HEL domains. However, theregion upstream of the papain-like protease domain in GLRaV-2 did notshow similarity to the corresponding region of BYV. In addition,variability in the residues located at the scissible bond (Gly in theBYV and Ala in the GLRaV-2) was present. Similar variability of thecleavage site residue in the P-PRO domain has been described in LChV(Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysisof Little Cherry Virus, a Mealybug-Transmissible Closterovirus. J.General Virology 78: 2067-71 (1997), which is hereby incorporated byreference).

Database searching with the deduced amino acid sequence of the ORF1a/1bencoded protein revealed a significant similarity to the MT, HEL andRdRP domains of the other closteroviruses. The region downstream of theP-PRO cleavage site showed a significant similarity (57.% identity in a266-residues alignment) to the putative methyltransferase domain of BYVand contained all the conserved motifs typical of positive-strand RNAviral type I MTs (FIG. 3B). The C-terminal portion of the ORF1a wasidentified as a helicase domain, the sequence of which showed a highsimilarity (57.1% identity in a 315-residues alignment) to the helicasedomain of BYV and contained the seven conserved motifs characteristic ofthe Superfamily I helicase of positive-strand RNA viruses (FIG. 3C)(Hodgman, “A New Superfamily of Replicative Proteins,” Nature 333: 22-23(1988); Koonin and Dolja, “Evolution and Taxonomy of Positive-strand RNAViruses: Implications of Comparative Analysis of Amino Acid Sequences,”Crit. Rev. in Biochem. and Mol. Biol. 28: 375-430 (1993), both of whichare hereby incorporated by reference).

ORF1b encoded a 460 amino acid polypeptide with a molecular mass of 52,486 Da, counting from the frameshifting site. Database searching withthe RdRP showed a significant similarity to the RDRP domains of positivestrand RNA viruses. Comparison of the RdRP domains of GLRaV-2 and BYVshowed the presence of the eight conserved motifs of RdRP (FIG. 3D).

As shown in FIG. 8, a tentative phylogenetic tree of the RdRP of GLRaV-2with respect to other closteroviruses shows that it is closely relatedto the monopartite closteroviruses BYV, BYSV, and CTV.

In closteroviruses, a +1 ribosomal frameshift mechanism has beensuggested to be involved in the expression of ORF1b as a large fusionprotein with ORF1a (Agranovsky et al., “Beet Yellows Closterovirus:Complete Genome Structure and Identification of a Papain-like ThiolProtease,” Virology 198: 311-24 (1994); Karasev et al., “CompleteSequence of the Citrus Tristeza Virus RNA Genome,” Virologv 208: 511-20(1995); Klaassen et al., “Genome Structure and Phylogenetic Analysis ofLettuce Infectious Yellows Virus, a Whitefly-Transmitted, BipartiteClosterovirus,” Virology 208: 99-110 (1995); Karasev et al.,“Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genomeand Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996); Jelkmann et al., “Complete Genome Structure andPhylogenetic Analysis of Little Cherry Virus, a Mealybug-TransmissibleClosterovirus,” J. General Virology 78: 2067-71 (1997), all of which arehereby incorporated by reference). In the overlapping ORF1a/1b region ofBYV, the slippery sequence of GGGUUUA and two hairpins structure(stem-loop and pseudoknot) are believed to result in a +1 frameshift(Agranovsky et al., “Beet Yellows Closterovirus: Complete GenomeStructure and Identification of a Papain-like Thiol Protease,” Virology198: 311-24 (1994), which is hereby incorporated by reference). None ofthese features are conserved in CTV and BYSV (Karasev et al., “CompleteSequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20(1995); Karasev et al., “Organization of the 3′-Terminal Half of BeetYellow Stunt Virus Genome and Implications for the Evolution ofClosteroviruses,” Virologv 221: 199-207 (1996), both of which are herebyincorporated by reference), in which a ribosomal pausing at a terminatoror at a rare codon was suggested to perform the same function.Comparisons of the nucleotide sequence of the C-terminal region of thehelicase and the N-terminal region of RdRP of GLRaV-2 with the sameregion of other closteroviruses revealed a significant similarity toBYV, BYSV, and CTV. As shown in FIG. 4, the terminator UAG at the end ofC′-terminal helicase of GLRaV-2 aligned with the terminator UAG of BYVand BYSV, and arginine CGG codon of CTV.

ORF2 encodes a small protein consisting of 171 bp (57 amino acid) with amolecular mass of 6,297 Da. As predicted, the deduced amino acidsequence includes a stretch of nonpolar amino acids, which is presumedto form a transmembrane helix. A small hydrophobic analogous protein isalso present in BYV, BYSV, CTV, LIYV, and LChV (Agranovsky et al.“Nucleotide Sequence of the 3′-Terminal Half of Beet YellowsClosterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J.General Virology 72: 15-24 (1991); Karasev et al., “Organization of the3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications forthe Evolution of Closteroviruses,” Virology 221: 199-207 (1996); Pappuet al., “Nucleotide Sequence and Organization of Eight 3′ Open ReadingFrames of the Citrus Tristeza Closterovirus Genome,” Virology 199: 35-46(1994); Klaassen et al., “Partial Characterization of the LettuceInfectious Yellows Virus Genomic RNAs, Identification of the CoatProtein Gene and Comparison of its Amino Acid Sequence With Those ofOther Filamentous RNA Plant Viruses,” J. General Virology 75: 1525-33(1994); Jelkmann et al., “Complete Genome Structure and PhylogeneticAnalysis of Little Cherry Virus, a Mealybug-TransmissibleClosterovirus,” J. General Virology 78: 2067-71 (1997), all of which arehereby incorporated by reference).

ORF3 encodes a 600 amino acid polypeptide with a molecular mass of65,111 Da, which is homologous to the HSP70 cellular heat shock protein.HSP70 is highly conserved among closteroviruses and is probably involvedin ATPase activity and the protein to protein interaction for chaperoneactivity (Agranovsky et al. “The Beet Yellows Closterovirus p65Homologue of HSP70 Chaperones has ATPase Activity Associated with itsConserved N-terminal Domain but Interact with Unfolded Protein Chains,”J. General Virology 78: 535-42 (1997); Agranovsky et al., “BacterialExpression and Some Properties of the p65, a Homologue of Cell HeatShock Protein HSP70 Encoded in RNA Genome of Beet YellowsClosterovirus,” Doklady Akademii Nauk. 340: 416-18 (1995); Karasev etal., “HSP70-Related 65-kDa Protein of Beet Yellows Closterovirus is aMicrotubule-Binding Protein,” FEBS Letters 304: 12-14 (1992), all ofwhich are hereby incorporated by reference). As shown in FIG. 5,alignment of the complete ORF3 of GLRaV-2 with HSP70 homolog of BYVrevealed the presence of the eight conserved motifs. The percentagesimilarity of the HSP70 between GLRaV-2 and that of BYV, BYSV, CTV,LIYV, and LChV is 47.8%, 47.2%, 38.6%, 20.9%, and 17.7%, respectively.

ORF4 encodes a 551 amino acid protein with a molecular mass of 63,349Da. Database searching with the ORF4 protein product did not identifysimilar proteins except those of its counterparts in closteroviruses,BYV (P64), BYSV (P61), CTV (P61), LIYV (P59), and LChV (P61). Thisprotein is believed to be a putative heat shock 90 protein. As shown inFIG. 9, two conserved motifs which were present in BYV (Agranovsky etal. “Nucleotide Sequence of the 3′-Terminal Half of Beet YellowsClosterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J.General Virology 72: 15-24 (1991), which is hereby incorporated byreference) and CTV (Pappu et al., “Nucleotide Sequence and Organizationof Eight 3′ Open Reading Frames of the Citrus Tristeza ClosterovirusGenome,” Virology 199: 35-46 (1994), which is hereby incorporated byreference) were also identified in the ORF4 of GLRaV-2.

ORF5 and ORF6 encode polypeptides with molecular mass of 24,803 Da and21,661 Da, respectively. The start codon for both ORFs is in a favorablecontext for translation. ORF6 was identified as the coat protein gene ofGLRaV-2 based on the sequence comparison with other closteroviruses. Thecalculated molecular mass of the protein product of ORF6 (21,662 Da) isin good agreement with the previously estimated 22˜26 kDa based onSDS-PAGE (Zimmermann et al., “Characterization and Serological Detectionof Four Closterovirus-like Particles Associated with Leafroll Disease onGrapevine,” J. Phytopathology 130: 205-18 (1990); Boscia et al.,“Nomenclature of Grapevine Leafroll-Associated PutativeClosteroviruses,” Vitis 34: 171-75 (1995), both of which are herebyincorporated by reference).

Database searching with the deduced amino acid sequence of the ORF6 ofGLRaV-2 showed a similarity with the coat proteins of closteroviruses,BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3. At the nucleotide level, thehighest percentage similarity was with the coat protein of BYSV (34.8%);at the amino acid level, the highest percentage similarity was with thecoat proteins of BYV (32.7%) and BYSV (32.7%). As shown in FIG. 6A,alignment of the amino acid sequence of the coat protein and coatprotein duplicate of GLRaV-2 with respect to other closterovirusesrevealed that the invariant amino acid residues (N. R. G. D.) werepresent in both ORF5 and ORF6 of GLRaV-2. Two of these amino acidresidues (R and D) are believed to be involved in stabilization ofmolecules by salt bridge formation and proper folding in the mostconserved core region of coat proteins of all filamentous plant viruses(Dolja et al., “Phylogeny of Capsid Proteins of Rod-Shaped andFilamentous RNA Plant Viruses Two Families With Distinct Patterns ofSequence and Probably Structure Conservation,” Virology 184: 79-86(1991), which is hereby incorporated by reference).

Identification of ORF6 as the coat protein gene was further confirmed byWestern blot following expression of a fusion protein, consisting of a22 kDa of ORF6 CP and a 42 kDa of maltose binding protein, produced bytransformed E. coli as described in Example 5 supra. As shown in FIG.6B, the putative phylogenetic tree of the coat protein and coat proteinduplicate of GLRaV-2 with those of other closteroviruses showed thatGLRaV-2 is more closely related to aphid transmissible closteroviruses(BYV, BYSV, and CTV) (Candresse, “Closteroviruses and Clostero-likeElongated Plant Viruses,” in Encyclopedia of Virology, pp. 24248,Webster and Granoff, eds., Academic Press, New York (1994), which ishereby incorporated by reference) than to whitefly (LIYV) or mealybugtransmissible closteroviruses (LChV and GLRaV-3) (Raine et al.,“Transmission of the Agent Causing Little Cherry Disease by the AppleMealybug Phenacoccus aceris and the Dodder Cuscuta Lupuliformis,”Canadian J. Plant Pathology 8: 6-11 (1986); Jelkmann et al., “CompleteGenome Structure and Phylogenetic Analysis of Little Cherry Virus, aMealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71(1997); Rosciglione and Gugerli, “Transmission of Grapevine LeafrollDisease and an Associated Closterovirus to Healthy Grapevine by theMealybug Planococcus ficus,” Phytoparasitica 17: 63 (1989); Engelbrechtand Kasdorf, “Transmission of Grapevine Leafroll Disease and AssociatedClosteroviruses by the Vine Mealybug planococcus-ficus,” Phytophlactica,22: 341-46 (1990); Cabaleiro and Segura, 1997; Petersen and Charles,“Transmission of Grapevine Leafroll-Associated Closteroviruses byPseudococcus longispinus and P. calceolariae. Plant Pathology 46: 509-15(1997), all of which are hereby incorporated by reference).

ORF7 and ORF8 encode polypeptides of 162 amino acid with a molecularmass of 18,800 Da and of 206 amino acid with a molecular mass of 23,659Da, respectively. Database searching with the ORF7 and ORF8 showed nosignificant similarity with any other proteins. Nevertheless, thesegenes were of similar in size and location as those observed in thesequence of other closteroviruses, BYV (P20, P21), BYSV (P18, P22), andLChV (P21, P27) (FIG. 7). However, conserved regions were not observedbetween the ORF7 or ORF8 and its counterparts in BYV, BYSV, and LChV.

The 3′ terminal untranslated region (3′-UTR) consists of 216nucleotides. Nucleotide sequence analysis revealed a long oligo(A) tractclose to the end of the GLRaV-2 genome which is similar to that observedin the genome of BYV and BYSV (Agranovsky et al. “Nucleotide Sequence ofthe 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome UniqueArrangement of Eight Virus Genes,” J. General Virology 72: 15-24 (1991);Karasev et al., “Organization of the 3′-Terminal Half of Beet YellowStunt Virus Genome and Implications for the Evolution ofClosteroviruses,” Virology 221: 199-207 (1996), both of which are herebyincorporated by reference). The genome of BYV ends in CCC, BYSV, and CTVends in CC with an additional G or A in the double-stranded replicativeform of BYSV (Karasev et al., “Organization of the 3′-Terminal Half ofBeet Yellow Stunt Virus Genome and Implications for the Evolution ofClosteroviruses,” Virology 221: 199-207 (1996), which is herebyincorporated by reference) and CTV (Karasev et al., “Complete Sequenceof the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995),which is hereby incorporated by reference), respectively. GLRaV-2 hadCGC at the 3′ terminus of the genome. Recently, a conserved 60 ntcis-element was identified in the 3′-UTR of three monopartiteclosteroviruses, which included a prominent conserved stem and loopstructure (Karasev et al., 1996). As shown in FIG. 10, alignment of the3′-UTR sequence of GLRaV-2 with the same regions of BYV, BYSV, and CTVshowed the presence of the same conserved 60 nt stretch. Besides thiscis-element, conserved sequences were not found in the 3′ UTRs ofGLRaV-2, BYV, BYSV, and CTV.

The closteroviruses studied so far (e.g., BYV, BYSV, CTV, LIYV, LChV,and GLRaV-3) have apparent similarities in genome organization, whichinclude replication associated genes that consist of MT, HEL, and RdRPconserved domains and a five-gene array unique for closteroviruses(Dolja et al. “Molecular Biology and Evolution of Closteroviruses:Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology32: 261-85 (1994); Agranovsky “Principles of Molecular Organization,Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv.in Virus Res. 47: 119-218 (1996); Jelkmann et al., “Complete GenomeStructure and Phylogenetic Analysis of Little Cherry Virus, aMealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71(1997); Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirdsof the Grapevine Leafroll Associated Virus-3 Genome Reveals a TypicalMonopartite Closterovirus,” J. General Virology 79(5): 1289-1301 (1998),all of which are hereby incorporated by reference).

The above data clearly shows that GLRaV-2 is a closterovirus. In thegenome of GLRaV-2, two putative papain-like proteases were identifiedand an autoproteolytic cleavage process was predicted. The replicationassociated proteins consisting of MT, HEL, and RdRP conserved motifswere also identified, which were phylogenetically closely related to thereplication associated proteins of other closteroviruses. A unique genearray including a small hydrophobic transmembrane protein, HSP70homolog, HSP90 homolog, diverged CP and CP was also preserved inGLRaV-2. In addition, the calculated molecular mass (21,661 Da) of thecoat protein (ORF6) of GLRaV-2 is in good agreement with that of theother closteroviruses (22 to 28 kDa) (Martelli and Bar-Joseph,“Closteroviruses: Classification and Nomenclature of Viruses,” FifthReport of the International Committee on Taxonomy of Viruses, Francki etal., eds., Springer-Verlag Wein, New York, p. 345-47 (1991); Candresseand Martelli, “Genus Closterovirus,” in Virus Taxonomy, Report of theInternational Committee on Taxonomy of Viruses, Murphy et al., eds.,Springer-Verlag., NY, p. 461-63 (1995), both of which are herebyincorporated by reference). Two ORFs downstream of the CP are ofsimilar, in size and location, to those observed in the genome of BYV.Furthermore, lack of a poly(A) tail at the 3′ end of GLRaV-2 is also ingood agreement with other closteroviruses. Like all otherclosteroviruses, the expression of ORF1b is suspected to occur via a +1ribosomal frameshift and the 3′ proximal ORFs are probably expressed viaformation of a nested set of subgenomic RNAs. Since the slipperysequence, stem-loop and pseudoknot structure involved in the frameshiftof BYV were absent in GLRaV-2, the +1 frameshift of GLRaV-2 might be thesame as proposed for CTV (Karasev et al., “Complete Sequence of theCitrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995), which ishereby incorporated by reference) and BYSV (Karasev et al.,“Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genomeand Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996), which is hereby incorporated by reference).

Overall, GLRaV-2 is more closely related to monopartite closterovirusesBYV, BYSV, and CTV than to GLRaV-3 (FIG. 7) (Ling et al., “NucleotideSequence of the 3′ Terminal Two-Thirds of the Grapevine LeafrollAssociated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,”J. General Virology 79(5): 1289-1301 (1998), which is herebyincorporated by reference), even though the latter causes similarleafroll symptoms in grapevine (Rosciglione and Gugerli, “Maladies del'Enroulement et du Bois Strie de la Vigne: Analyse Microscopique etSerologique (Leafroll and Stem Pitting of Grapevine: Microscopical andSerological Analysis),” Rev Suisse Viticult Arboricult Horticulture 18:207-11 (1986); Hu et al., “Characterization of Closterovirus-LikeParticles Associated with Grapevine Leafroll Disease,” J. Phytopathology128: 1-14 (1990), both of which are hereby incorporated by reference).

Closteroviruses are a diverse group with complex and heterogeneousgenome organizations. So far, GLRaV-2 is the only closterovirus thatmatches with the genome organization of BYV, the type member of thegenus Closterovirus. In addition, the genomic RNA of GLRaV-2 is aboutthe same size as that of BYV; however, the transmission vector ofGLRaV-2 is unknown. The genome organization of GLRaV-2 is more closelyrelated to the aphid transmissible closteroviruses (BYV and CTV) than towhitefly (LIYV) or mealybug transmissible closteroviruses (LChV andGLRaV-3). Thus, it is possible that GLRaV-2 is transmitted by aphids.Aphid transmission experiments with GLRaV-2 should provide informationthat might help develop methods for further control of GLRaV-2.

A total of 15,500 nucleotides or over 95% of the estimated GLRaV-2genome has been cloned and sequenced. GLRaV-2 and GLRaV-3 (Ling et al.,“Nucleotide Sequence of the 3′ Terminal Two-Thirds of the GrapevineLeafroll Associated Virus-3 Genome Reveals a Typical MonopartiteClosterovirus,” J. General Virology 79(5): 1289-1301 (1998), which ishereby incorporated by reference) are the first grapevine leafrollassociated closteroviruses that have been almost completely sequenced.The above data clearly justify the inclusion of GLRaV-2 into the genusClosterovirus. In addition, the information regarding the genome ofGLRaV-2 would provide a better understanding of this and related GLRaVs,and add fundamental knowledge to the group of closteroviruses.

Example 7 Construction of the CP Gene of GLRaV-2 in Plant ExpressionVector

GLRaV-2 infected Vitis vinifera, cv Pinot Noir grapevines originatedfrom a vineyard in central New York was used as the virus isolate, fromwhich the cp gene of GLRaV-2 was identified. Based on the sequenceinformation, two oligonucleotide primers have been designed. The senseprimer CP-96F (SEQ. ID. No. 21) starts from the ATG initiation codon ofthe coat protein gene and the complementary primer CP-96R (SEQ. ID. No.22) starts from 56 nucleotides downstream of the stop codon of the CPgene. A Nco I restriction site (11 bp in SEQ. ID. No. 21 and 13 bp inSEQ. ID. No. 22) is introduced in the beginning of both primers tofacilitate the cloning. The coat protein gene of GLRaV-2 was amplifiedfrom dsRNA extracted from GLRaV-2 infected grapevine using reversetranscriptase polymerase chain reaction (RT-PCR). The PCR-amplified CPproduct was purified from low melting temperature agarose gel, digestedwith Nco I and cloned into the same enzyme digested plant expressionvector pEPT8 (shown at FIG. 11). After screening, the orientation ofrecombinant construct was checked by using the internal restriction siteof the CP gene and directly sequencing the CP gene. The recombinantconstruct with translatable (sense) full length coat protein gene,pEPT8CP-GLRaV2, was going through for the further cloning. The plantexpression cassette, which consisted of a double cauliflower mosaicvirus (CaMV) 35S-enhancer, a CaMV 35S-promoter, an alfalfa mosaic virus(ALMV) RNA4 5′ leader sequence, a coat protein gene of GLRaV-2(CP-GLRaV-2), and a CaMV 35S 3′ untranslated region as a terminator, wascut using the EcoR I restriction enzyme, isolated from low melting pointtemperature agarose gel, and cloned into the same restriction enzymetreated binary vector pGA482GG or pGA482G (a derivative of pGA482 (An etal., “Binary Vectors,” in Plant Molecular Biology Manual, pp. A3: 1-19,Gelvin and Schilperoot, eds., Kinwer Academic Publishers, Dordrecht,Netherlands (1988), which is hereby incorporated by reference). Theresulting recombinants constructs are pGA482GG/EPT8CP-GLRaV2 (shown atFIG. 11A), which contain both neomycin phosphotransferase (npt II) andP-glucuronidase (GUS) at the internal region of the T-DNA, andpGA482G/EPT8CP-GLRaV2 (shown at FIG. 11B) without GUS. Theserecombinants constructs were separately introduced by electroporationinto disarmed avirulent Agrobacterium tumefaciens strain C58Z707. TheAgrobacterium tumefaciens containing the vector was used to infectNicotiana benthamiana wounded leaf disks according to the procedureessentially described by Horsch et al., “A Simple and General Method forTransferring Genes into Plants,” Science 277: 1229-1231 (1985), which isincorporated herein by reference.

Example 8 Analysis of Transgenic Nicotiana benthamiana Plants with theCP Gene of GLRaV-2

NPT II-ELISA: Double-antibody sandwich enzyme linked immuosorbent assay(DAS-ELISA) was used to detect the npt II enzyme with an NPT II-ELISAkit (5′ prime to 3′ prime, Inc., Boulder, Co.).

Indirect ELISA: Polyclonal antibodies to GLRaV-2, which were preparedfrom the coat protein expressed in E. coli, were used. Plates werecoated with homogenized samples in extraction buffer (1:10, w/v)(phosphate buffered saline containing 0.05% Tween 20 and 2% polyvinylpyrrolidone) and incubated overnight at 4° C. After washing withphosphate buffered saline containing 0.05% Tween 20 (PBST), the plateswere blocked with blocking buffer (phosphate buffered saline containing2% BSA) and incubated at room temperature for 1 hr. The anti-GLRaV-2 IgGwas added at 2 μg/ml after washing with PBST. After incubation at 30 Cfor 4 hr, the plates were washed with PBST, and the goat anti-rabbit IgGconjugate of alkaline phosphotase (Sigma) was added at 1:10,000dilution. The absorbance was measured at 405 nm with a MicroELISAAutoReader. In addition, Western blot was also performed according tothe method described by Hu et al., “Characterization ofClosterovirus-like Particle Associated Grapevine Leafroll Disease,” J.Phytophathology 128: 1-14, (1990), which is incorporated herein byreference.

PCR analysis: Genomic DNA was extracted from leaves of putativetransgenic and non-transgenic plants according to the method describedby Cheung et al., “A Simple and Rapid DNA Microextraction Method forPlants, Animal, and Insect Suitable for RAPD and other PCR analysis,”PCR Methods and Applications 3: 69 (1996), which is incorporated hereinby reference. The extracted total DNA served as the template for PCRreaction. The primers CP-96F and CP-96R (SEQ. ID. Nos. 21 and 22,respectively) for the CP gene of GLRaV-2, as well as npt II 5′- and3′-primers were used for PCR analysis. PCR reaction was performed at the94° C.×3 min for one cycle, followed by 30 cycles of 94° C.×1 min, 50°C.×1 min, and 72° C.×2:30 min with an additional extension at 72° C. for10 min. The PCR product was analyzed on agarose gel.

After transformation, a total of 42 kanamycin resistant Nicotianabenthamiana lines (R₀) were obtained, of which the leaf samples weretested by NPT II enzyme activity. Among them, 37 lines were NPT IIpositive by ELISA, which took about 88.0% of total transformants.However, some of NPT II negative plants were obtained among theseselected kanamycin resistant plants. All of the transgenic plants wereself-pollinated in a greenhouse, and the seeds from these transgeniclines were germinated for further analysis.

The production of GLRaV-2 CP in transgenic plants was detected byindirect ELISA prior to inoculation, and the results showed that GLRaV-2CP gene expression was not detectable in all transgenic plants tested.This result was further confirmed with Western blot. Using the antibodyto GLRaV-2, the production of the CP was not detected in the transgenicand nontransgenic control plants. However, a protein of expected size(˜22 kDa) was detected in GLRaV-2 infected positive control plants. Thisresult was consistent with the ELISA result. The presence of the CP geneof GLRaV-2 in transgenic plants was detected from total genomic DNAextracted from plants tissue by PCR analysis (FIG. 12). The DNA productof expected size (653 bp) was amplified from twenty tested transgeniclines, but not in non-transgenic plants. The result indicated that theCP gene of GLRaV-2 was present at these transgenic lines, which was alsoconfirmed by Northern blot analysis.

Example 9 R₁ and R2 transgenic Nicotiana benthamiana Plants AreResistant to GLRaV-2

Inoculation of transgenic plants: GLRaV-2 isolate 94/970, which wasoriginally identified and transmitted from grapevine to Nicotianabenthamiana in South Africa (Goszczynski et al., “Detection of TwoStrains of Grapevine Leafroll-Associated Virus 2,” Vitis 35: 133-35(1996), which is incorporated herein by reference), was used asinoculum. The CP gene of isolate 94/970 was sequenced; and it isidentical to the CP gene used in construction. Nicotiana benthamiana isan experimental host of GLRaV-2. The infection on it produces chloroticand occasional necrotic lesions followed by systemic vein clearing. Thevein clearing results in vein necrosis. Eventually the infected plantsdied, starting from the top to the bottom.

At five to seven leaf stage, two youngest apical leaves were challengedwith GLRaV-2 isolate 94/970. Inoculum was prepared by grinding 1.0 gGLRaV-2 infected Nicotiana benthamiana leaf tissue in 5 ml of phosphatebuffer (0.01 MK2HPO₄, PH7.0). The tested plants were dusted withcarborundum and rubbed with the prepared inoculum. Non-transformedplants were simultaneously inoculated as above. The plants were observedfor symptom development every other day for 60 days after inoculation.Resistant R1 transgenic plants were carried on to R2 generation forfurther evaluation.

Transgenic progenies from 20 R₀ lines were initially screened for theresistance to GLRaV-2 followed by inoculation with GLRaV-2 isolate94/970. The seedlings of the transgenic plants (NPT II positive), andnontransformed control plants were inoculated with GLRaV-2. Afterinoculation, the reaction of tested plants were divided into threetypes: highly susceptible (i.e. typical symptoms were observed two tofour weeks postinoculation); tolerant (i.e. no symptom was developed inthe early stage and typical symptoms was shown four to eight weekspostinoculation); and resistant (i.e. the plants remained asymptomaticeight weeks postinoculation). Based on the plant reaction, the resistantplants were obtained from fourteen different lines (listed in Table 1below). In each of these fourteen lines, there was no virus detectedwithin these plants by ELISA at 6 weeks postinoculation. In contrast,GLRaV-2 was detected in symptomatic plants by indirect ELISA. In theother six lines, although there were a few plants with some kind ofdelay in symptom development, all the inoculated transgenic plants diedat three to eight weeks postinoculation. Based on the initial screeningresults, five representative lines consisting of three resistant lines(1, 4, and 19) and two susceptible lines (12 and 13) were selected forthe further analysis. TABLE 1 Reaction of Tested Plants No. Line No. HST HR line 1 39 14 3 22 line 2 36 7 6 23 line 3 38 11 4 23 line 4 31 4 522 line 5 33 6 13 14 line 6 36 4 16 16 line 7 32 5 9 18 line 8 37 22 9 6line 9 36 9 12 15 line 10 14 13 1 0 line 11 13 11 2 0 line 12 17 16 1 0line 13 16 14 0 0 line 14 17 17 0 0 line 15 32 30 2 0 line 16 33 6 13 14line 17 12 0 1 11 line 19 15 0 0 15 line 20 19 3 0 16 line 21 14 1 3 10control 15 15 0 0No Line: include transgenic lines and nontransformed control;No: the number of transgenic and nontransformed plants;HS: highly susceptible, typical symptoms were observed two to four weeksafter inoculation;T: tolerant, the symptoms were observed five to eight weeks afterinoculation; andHR: plants remain without asymptoms after eight weeks inoculation.

Table 2 below shows the symptom development in transgenic plantsrelative to non-transgenic control plants in the five selected lines inseparate experiments. Non-transgenic control plants were all infectedtwo to four weeks after inoculation, which showed typical GLRaV-2symptoms on Nicotiana benthamiana, including chlorotic and local lesionsfollowed by systemic vein clearingand vein necrosis on the leaves. Threeof the tested lines (1, 4, and 19) showed some resistance that wasmanifested by either an absence or a delay in symptom development. Twoother lines, 12 and 13, developed symptoms at nearly the same time asthe non-transformed control plants. From top to bottom, the leaves ofinfected plants gradually became yellow, wilted, and dried, and,eventually, the whole plants died. No matter when infection occurred,the eventual result was the same. Six weeks after inoculation, allnon-transgenic plants and the susceptible plants were dead. Sometolerant plants started to die. In contrast, the asymptomatic plantswere flowering normally and pollinating as the non-inoculated healthycontrol plants (FIG. 13). TABLE 2 Reaction of Tested Plants No. Line No.HS T HR line 1 19 5 6 8 line 4 15 9 1 5 line 12 16 14 2 0 line 13 18 135 0 line 19 13 10 0 3 non-transgenic 24 23 1 0No. Line: include transgenic lines and nontransformed control;No.: Number of transgenic and nontransformed plants tested;HS: highly susceptible; typical symptoms were observed two to four weeksafter inoculation;T: tolerant, the symptoms were observed five to eight weekspostinoculation; andHR: plants remain without asymptoms after eight weeks inoculation.

ELISA was performed at 6 weeks postinoculation to test the GLRaV-2replication in the plants. Presumably, the increased level of CPreflected virus replication. The result showed that the absorbance valuein symptomatic plants reached (OD) 0.7 to 3.2, compared to (OD)0.10-0.13 prior to inoculation. In contrast, GLRaV-2 was not detected inasymptomatic plants, of which the absorbance value was the same ornearly the same as that of healthy nontransformed control plants. Thedata confirmed that virus replicated in symptomatic plants, but not inasymptomatic plants. The replication of GLRaV-2 was suppressed inasymptomatic plants. This result implicated that another mechanism otherthan the CP-mediated resistance was probably involved.

Three R2 progenies derived from transgenic resistant plants of lines 1,4, and 19 were generated and utilized to examine the stable transmissionand whether resistance was maintained in R2 generation. These resultsare shown in Table 3 below. NPT II analysis revealed that R2 progenywere still segregating. The CP expression in R2 progeny was stillundetectable. After inoculation, all the nontransgenic plants wereinfected and showed GLRaV-2 symptoms on the leaves after 24 dayspostinoculation. In contrast, the inoculated transgenic R₂ progenyshowed different levels of resistance from those highly susceptible tohighly resistant. The tolerant and resistant plants were manifested by adelay in symptom development and absence of symptoms, respectively. At 6weeks postinoculation, GLRaV-2 was detected in the tolerant symptomaticinfected plants by indirect ELISA; but not in asymptomatic plants. Thisresult indicated that virus replication was suppressed in theseresistant plants, which was confirmed by Western blot. These resistantplants remained asymptomatic eight weeks postinoculation, and they wereflowering normally and pollinating. TABLE 3 Reaction of NPT II TestedPlants No. Line No. Plants positive/negative HS T HR line 1/22 12 12/203 3 6 line 1/30 11 8/3 7 2 2 line 1/31 11 10/1  6 3 2 line 1/35 10 10/0 4 6 0 line 1/41 8 7/1 2 2 4 line 4/139 12 11/1  4 4 3 line 4/149 10 7/34 5 1 line 4/152 10 8/2 9 0 1 line 4/174 9 8/1 4 0 4 line 19/650 1110/1  7 0 2 line 19/657 12 12/0  6 2 4 line 19/659 12 8/4 5 2 5 line19/660 10 8/2 3 6 1 non-transformed 12  0/12 12 0 0 CKHS: highly susceptible, typical symptoms were observed two to four weeksafter inoculation;T: tolerant, the symptoms were observed five to eight weekspostinoculation; andHR: plants remain asymptomatic at eight weeks postinoculation.

Example 10 Evidence for RNA-Mediated Protection in Transgenic Plants

Northern blot analysis: Total RNA was extracted from leaves prior toinoculation following the method described by Napoli et al., Plant Cell2: 279-89 (1990), which is hereby incorporatd by reference. Theconcentration of the extracted RNA was measured by spectrophotometer atOD 260. About 10 g of total RNA was used for each sample. The probe usedwas the 3′ one third of GLRaV-2 CP gene, which was randomly labeled with³²P (α-dATP) using Klenow fragment of DNA polymerase I.

Using a DNA corresponding to the 3′ one third CP gene sequence as probe,a single band was detected in the RNA extracted from susceptible plantsfrom R1 progeny of lines 5, 12, and 13 by Northern hybridization. Therewas little or no signal detected in the transgenic plants from R1progeny of line 1, 4, and 19. This RNA is not present in nontransformedcontrol plants. The size of the hybridization signal was estimated to anapproximately 0.9 kb nucleic acid, which was about the same as estimated(FIG. 14). In lines of 1, 4, and 19, the steady state level of RNAexpression was also low in R2 progeny. This data showed that susceptibleplants from lines 12 and 13 had high mRNA level and all transgenicplants from lines 1, 4, and 19 had low mRNA level.

Example 11 Transformation and Analysis of Transgenic Grapevines with theCP Gene of GLRaV-2

Plant materials: The rootstock cultivars Couderc 3309 (3309C) (V.riparia×V rupestris), Vitis riparia ‘Gloire de Montpellier’ (Gloire),Teleki 5C (5C) (V. berlandieri×V. riparia), Millardet et De Grasset101-14 (101-14 MGT) (V. riparia×V. rupestris), and Richter 110 (10R) (V.rupestris×V. berlandieri) were utilized. Initial embryogenic calli ofGloire were provided by Mozsar and Süle (Plant Protection Institute,Hungarian Academy of Science, Budapest). All other plant materials camefrom a vineyard at the New York State Agricultural Experiment Station,Geneva, N.Y. Buds were removed from the clusters and surface sterilizedin 70% ethanol for 1-2 min. The buds (from the greenhouse and the field)were transferred to 1% sodium hypochlorite for 15 min, then rinsed threetimes in sterile, double-distilled water. Anthers were excisedaseptically from flower buds with the aid of a stereo microscope. Thepollen was crushed on a microscope slide under a coverslip with a dropof acetocarmine to observe the cytological stage. This was done todetermine which stage was most favorable for callus induction.

Somatic embryogenesis and regeneration: Anthers were plated underaseptic conditions at a density of 40 to 50 per 9 cm diameter Petri dishcontaining MSE. Plates were cultured at 28° C. in the dark. Callus wasinitiated, and, after 60 days, embryos were induced and were transferredto hormone-free HMG medium for differentiation. Torpedo stage embryoswere then transferred from HMG to MGC medium to promote embryogermination. Cultures were maintained in the dark at 26-28° C. andtransferred to fresh medium at 3-4 week intervals. Elongated embryoswere transferred to rooting medium in baby food jars (5-8 embryos perjar). The embryos were grown in a tissue culture room at 25° C. with adaily 16 h photoperiod (76:mol. s) to induce shoot and root formation.After plants developed roots, they were transplanted to soil in thegreenhouse.

Transformation: The protocols used for transformation were modified fromthose described by Scorza et.al., “Transformation of Grape (Vitisvinifera L.) Zygotic-derived Somatic Embryos and Regeneration ofTransgenic Plants,” Plant Cell Rpt. 14: 589-92 (1995), which is herebyincorporated by reference. Overnight cultures of Agrobacterium strainC58Z707 or LBA4404 were grown in LB medium at 28° C. in a shakingincubator. Bacteria were centrifuged for 5 min at 3000-5000 rpm andresuspended in MS liquid medium (OD 1.0 at A600 nm). Calli with embryoswere immersed in the bacterial suspension for 15-30 min blotted dry, andtransferred to HMG medium with or without acetosyringone (100 μM).

Embryogenic calli were co-cultivated with the bacteria for 48 h in thedark at 28° C. Then, the plant material was washed in MS liquid pluscefotaxime (300 mg/ml) and carbenicillin (200 mg/ml) 2-3 times. Toselect transgenic embryos, the material was transferred to HMG mediumcontaining either 20 or 40 mg/L kanamycin, 300 mg/L cefotaxime, and 200mg/L carbenicillin. Alternatively, after co-cultivation, embryogeniccalli were transferred to initiation MSE medium containing 25 mg/lkanamycin plus the same antibiotics listed above. All plant materialswere incubated in continuous dark at 28° C. After growth on selectionmedium for 3 months, embryos were transferred to HMG or MGC withoutkanamycin to promote elongation of embryos. They were then transferredto rooting medium without antibiotics. Nontransformed calli were grownon the same media with and without kanamycin to verify the efficiency ofthe kanamycin selection process.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. An isolated RNA molecule encoding protein or polypeptide of a grapevine leafroll virus (type 2).
 2. The isolated RNA molecule according to claim 1, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
 3. An isolated DNA molecule encoding a protein or polypeptide of a grapevine leafroll virus (type 2).
 4. The isolated DNA molecule according to claim 3, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
 5. An expression system comprising a DNA molecule according to claim 3 in a-vector heterologous to the DNA molecule.
 6. The expression system according to claim 5, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
 7. A host cell transformed with a heterologous DNA molecule according to claim
 3. 8. The host cell according to claim 7, wherein the host cell is selected from the group consisting of Agrobacterium vitis and Agrobacterium tumefaciens.
 9. The host cell according to claim 7, wherein the host cell is selected from a group consisting of a grape cell, a citrus cell, a beet cell, and a tobacco cell.
 10. The host cell according to claim 7, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA-polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
 11. A transgenic plant cultivar comprising the DNA molecule according to claim
 3. 12. The transgenic plant cultivar according to claim 11, wherein the plant cultivar is selected from a group consisting of a grape plant cultivar, a citrus plant cultivar, a beet plant cultivar, and a tobacco plant cultivar.
 13. The transgenic plant cultivar according to claim 11, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
 14. A method of imparting grapevine leafroll virus resistance to a Vitis scion or rootstock cultivar or a Nicotiana cultivar comprising the steps of: (a) transforming of cells of a Vitis scion or rootstock cultivar or cells of a Nicotiana cultivar with an isolated DNA molecule encoding a protein or polypeptide of a grapevine leafroll virus (type 2); and (b) regenerating a Vitis scion or rootstock cultivar or a Nicotiana cultivar from said transformed cells.
 15. The method according to claim 14, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, and a coat protein.
 16. The method according to claim 14, wherein the protein or polypeptide is a grapevine leafroll virus (type 2) coat protein.
 17. The method according to claim 14, wherein the protein or polypeptide is a grapevine leafroll virus (type 2) heat shock 70 protein.
 18. The method according to claim 14, wherein the grapevine leafroll virus is GLRaV-2.
 19. The method according to claim 14, wherein said transforming is Agrobacterium mediated.
 20. The method according to claim 14, wherein said transforming comprises: propelling particles at grape or tobacco plant cells under conditions effective for the particles to penetrate into the cell interior; and introducing an expression vector comprising the DNA molecule into the cell interior. 